Functional, segregated, charged telodendrimers and nanocarriers and methods of making and using same

ABSTRACT

Provided are multiply functional charged telodendrimers. The telodendrimers can be used for protein encapsulation and delivery. The charged telodendrimers may have one or more crosslinking groups (e.g., boronic acid/catechol reversible crosslinking groups). The telodendrimers can aggregate to form nanoparticles. Cargo such as combinations of proteins and other materials may be sequestered in the core of the nanoparticles via non-covalent or covalent interactions with the telodendrimers. Such nanoparticles may be used in protein delivery applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional applicationSer. No. 15/759,665, filed on Mar. 13, 2018, which is a National Phaseof International Patent Application No. PCT/US2016/051266, filed on Sep.12, 2016, which claims priority to U.S. Provisional Application No.62/217,951, filed on Sep. 13, 2015, the disclosure of which is herebyincorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under contract no.1R01CA140449 awarded by the National Institutes of Health and NationalCancer Institute. The government has certain rights in the invention.

FIELD OF THE DISCLOSURE

The disclosure generally relates to telodendrimers. More particularlythe disclosure generally relates to functional, segregated, chargedtelodendrimers.

BACKGROUND OF THE DISCLOSURE

Protein therapy is, in a manner, limited by the lack of efficientnanocarriers for intracellular delivery while maintaining proteinbioactivity. A rational strategy to create small nanoparticles with highprotein loading ability and cell-penetration property is desired but isoften overlooked.

Currently, more than 130 bioactive proteins have been approved to treathuman diseases. The majority of these protein therapeutics target thereceptors or antigens expressed on the plasma membrane, such as insulinand antibodies. The modification of the pharmacokinetic of the proteinsby delivery system is able to enhance their therapeutic efficacy.PEGylation of protein has a long-standing history to efficiently prolongcirculation time, increase stability and reduce the immunogenicity ofprotein therapeutics, especially for recombinant protein therapeutics.Physical encapsulation of proteins into nano- or micro-particles hasbeen intensively studied for systemic or local administration. It isimportant to maintain protein structure and activity in such proteinencapsulation process, especially for the process involvinglyophilization or organic solvent applications. For example, the usageof organic solvents in the oil/water emulsion technique for theencapsulation of proteins into biodegradable polymeric microparticles,e.g., polylactic acid and polycaprolactone, usually causes thedenaturation of proteins with at least partial losses of activity.Encapsulation of proteins in aqueous environments, such as in hydrogelsand nanogels, represents a better way to sustain protein structure andactivities. However, these processes mostly relay on polymerization orchemical reactions to crosslink hydrogels at bulky scale or within thenanodispersed aggregates. The chemical process may lead to thecomplication in control of the physical properties, and the chemicalsused in these reactions may present as toxic impurity that hindersapplication in vivo. Efficient encapsulation of proteins in situ inbiologically relevant environments, e.g., pH, temperature and ionstrength without extra chemicals or steps needed are highly demanded forclinical development of protein therapeutics.

Even more proteins are possible to be therapeutics if they can bedelivered across plasma membrane into intracellular space, such asantibodies against intracellular proteins used in biochemistry assays orpathology detections. However, such exogenous proteins, even someendogenous proteins are not cell permeable by themselves due to theirsurface charge distributions, large molecular weights and vulnerabletertiary structures. In addition, they do not have receptors to mediatetheir intracellular uptake, which renders these proteins inactive.Therefore, the ability to create efficient vehicles for intracellularprotein delivery in vivo will expand the horizon dramatically indevelopment and application of therapeutic proteins in diseasetreatments. The recombinant proteins with targeting domains presentsolutions for intracellular delivery of such functional proteins.However, the tedious recombinant design/production and the costlyprocess for protein humanization hinder the development of suchrecombinant therapeutics. Cell-penetrating peptides and cationicpolymers/liposomes have been widely studied over the past few decadesfor intracellular delivery of biomacromolecules, such as genes andproteins while maintaining the bioactivity. However, the advancement ofthese vehicles are mainly hindered by their positive surface charges,that usually cause high cytotoxicity and are also subjected tononspecific phagocytosis by the reticuloendothelium systems in vivo.Polymeric vehicles hold great promise to overcome these shortages. Theapplication of microparticles and hydrogels for intracellular proteindelivery is limited by their large sizes. The delivery systems based onnano-scaled vehicles are highly promising for intracellular delivery ofprotein therapeutics to treat human diseases, especially for cancers.

A recent study by Farokhzad and coworkers showed great promise tominimize zeta potential of the cationic nanocarriers bypost-modification of the protein-conjugated nanoparticles withlipid-polyethylene glycol, yielding multinuclear nanoparticles withdiameters of 100-150 nm. The protein aggregation and dehydration maylikely occur within the big aggregates, which may be irreversible andpotentially leads to protein denaturation. In addition, many studiessuggested that small particle sizes (10-30 nm) are beneficial fortherapeutic delivery with large volume intratumoral distribution anddeep tumor penetration. Coating protein with a layer of polymer inaqueous solution is able to address all these concerns to avoid proteinaggregation, dehydration and form small particle sizes similar topolymeric micelles (10-30 nm). Optimization of the information encodedin macromolecular building blocks is able to tune the sizes ofself-assembled nanoparticles. In a previous study, we observed that theprecise control on macromolecular architecture and composition wascritical to optimize the particle sizes and drug loading behaviors,which seriously affected the colon cancer treatment efficiency.

SUMMARY OF THE DISCLOSURE

In an aspect, the present disclosure provides charged telodendrimers.The charged telodendrimers are linear-dendritic copolymers. The chargedtelodendrimers are functional segregated telodendrimers having, forexample, two or three functional segments. In an embodiment, thefunctional segments are a hydrophilic segment and a hydrophobic segment.The hydrophilic segment comprises one or more charged groups. Thecharged telodendrimers may comprise an intermediate layer. The chargedtelodendrimers may have one or more crosslinking groups (e.g., boronicacid/catechol reversible crosslinking groups). The chargedtelodendrimers may comprise PEG groups that can form a PEG layer. In anembodiment, the present disclosure provides charged telodendrimers thatare functional and spatially segregated telodendrimers having 1 to 128charged groups. The telodendrimers may have one or more crosslinkinggroups (e.g., reversible boronate crosslinking groups/reversiblecatechol crosslinking groups). In an embodiment, the telodendrimers arefunctional segregated telodendrimers having three functional segments.In various examples, a charged telodendrimer has one or more feature ofthe charged telodendrimers of Statements 1 to 15 or a combinationthereof. The telodendrimers may be used to stabilize proteins. The typeof charge, the number of charged groups, the ratio of charged groups tohydrophobic groups (if present), the spatial orientation of the chargedgroups, and/or the density of the charged groups can be selected tostabilize a specific protein.

In an aspect, the present disclosure provides nanocarriers comprisingcharged telodendrimers of the present disclosure. In an embodiment, acomposition comprises an aggregate of a plurality of the telodendrimersthat form a nanocarrier having a hydrophobic core and a hydrophilicexterior. In various examples, a nanocarrier has one or more feature ofthe nanocarriers of Statements 16 to 18, or a combination thereof. Thenanocarrier may be a telodendrimer micelle. A telodendrimer micelle is ananoconstruct formed by the self-assembly of the telodendrimer inaqueous solution. The telodendrimer micelle can serve as a nanocarrierto load various types of proteins. In an embodiment, the nanocarriercomprises a plurality of charged telodendrimer compounds. In anembodiment, the nanocarrier comprises one or more charged proteins. Thenanocarriers comprising one or more charged proteins may have a diameterof 5 nm to 50 nm, including all integer nm values and rangestherebetween. In an embodiment, the nanocarriers comprising one or morecharged proteins may have a diameter of 10 nm to 30 nm. Thetelodendrimers can be designed such that each of the proteins carriedwill have a different release profile. Examples of conditions that canaffect the release profile of carried proteins include time andbiological environment.

The charged telodendrimers can be present in a composition. In anembodiment, the composition comprises one or more chargedtelodendrimers. The composition may comprise a mixture of positivelycharged telodendrimers, a mixture of negatively charged telodendrimers,or a mixture of positively and negatively charged telodendrimers. In anembodiment the composition further comprises one or more proteins. In anembodiment the composition further comprises one or more drugs. Thecomposition can have a formulation as disclosed herein. For example, thecomposition can be a pharmaceutical composition as described herein.

In an aspect, the present disclosure provides methods of using chargedtelodendrimers of the present disclosure. The telodendrimers can beused, for example, in methods of treatment. The compositions ornanocarriers of the present disclosure can be used to treat any diseaserequiring the administration of a protein, such as, for example, bysequestering a protein in the interior of the nanocarrier, anddelivering said protein to a target. The protein(s) can be deliveredsystemically or intracellularly. In an embodiment, compositionscomprising the telodendrimers are used in a method for treating adisease. In some embodiments, the present disclosure provides a methodof treating a disease, including administering to a subject in need ofsuch treatment a therapeutically effective amount of a composition ornanocarrier of the present disclosure, where the nanocarrier includes anencapsulated protein. The pharmaceutical preparations are typicallydelivered to a mammal, including humans and non-human mammals. Non-humanmammals treated using the present methods include domesticated animals(e.g., canine, feline, murine, rodentia, and lagomorpha) andagricultural animals (e.g., bovine, equine, ovine, and porcine). Inpracticing the methods of the present disclosure, the pharmaceuticalcompositions can be used alone, or in combination with other therapeuticor diagnostic agents.

In an aspect, compositions or nanocarriers comprising chargedtelodendrimers are used in imaging methods. In an embodiment, acomposition or nanocarrier comprises an imaging agent. In an embodiment,the present disclosure provides a method of imaging, includingadministering to a subject to be imaged, an effective amount of acomposition or nanocarrier of the present disclosure, wherein thecomposition or nanocarrier includes an imaging agent. In otherembodiments, the method of treating and the method of imaging areaccomplished simultaneously using a nanocarrier having a therapeuticprotein, and/or an imaging agent-labeled protein.

DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the nature and objects of the disclosure,reference should be made to the following detailed description taken inconjunction with the accompanying figures.

FIG. 1. Hypothetical assembly models of protein-telodendrimer complex.

FIG. 2. Loading ability of telodendrimers containing 4 or 8 guanidinegroups with C17, CHO or VE hydrophobic groups for FITC-BSA determined byan agarose gel retention assay. The feed mass ratio is 2/1 (P/T).

FIG. 3. Calorimetric titration of PEG^(5k)(ArgArg-L-C17)₄ (a),PEG^(5k)(ArgArg-L-CHO)₄ (b), and PEG^(5k)(ArgArg-L-VE)₄ (c) with BSA at37° C. in PBS (1×)_(.)

FIG. 4. In vitro binding of telodendrimer to protein measured by BLI.(a) Schematic illustration of the association in telodendrimer solution(left) and dissociation in BSA solution (right) for thestreptavidin-coated biosensors prewetted with BSA solution. (b) Kineticsfor association in PEG^(5k)(ArgArg-L-C17)₄ solution (500 nM) anddissociation in PBS and BSA solutions of different concentrations. (c)Dissociation rate constants determined by fitting the curves in (b).(d-f) Kinetics for association in PEG^(5k)(ArgArg-L-C17)₄ (d),PEG^(5k)(ArgArg-L-CHO)₄ (e), and PEG^(5k)(ArgArg-L-VE)₄ (f) solutions(75-600 nM) and dissociation in BSA solutions (40 mg/mL).

FIG. 5. Determination of the roles of charged and hydrophobic moietiesin telodendrimers for protein binding. (a) Loading ability of differenttelodendrimers for FITC-BSA determined by an agarose gel retentionassay. The feed mass ratio of is 1/3 (P/T). (b-d) Kinetics forassociation in PEG^(5k)(Arg-L-CHO)₄ (b), PEG^(5k)Arg₄AA₄ (c), andPEG^(5k)(Arg(Pbf)-L-CHO)₄ (d) solutions (75-600 nM) and dissociation inBSA solution (40 mg/mL) measured by BLI.

FIG. 6. CLSM images of HT-29 cells incubated at 37° C. for 3 h with freeFITC-BSA (a), and FITC-BSA loaded in the nanoparticles of telodendrimerscontaining four (b) or eight (c) guanidine groups, and C17, CHO or VE ashydrophobic groups at a P/T ratio of 1/3 by weight. The images weretaken at a magnification of 60×. The cell nuclei were stained with DAPI(blue).

FIG. 7. Cell viability assay on U87 cells after a 72 h continuousincubation at 37° C. for free DT₃₉₀, and DT₃₉₀-loaded telodendrimernanoparticles.

FIG. 8. Synthetic route for telodendrimers with four guanidine groups.

FIG. 9. Synthetic route for telodendrimers with eight guanidine groups.

FIG. 10. Chemical structures of telodendrimers containing guanidinegroups and/or cholesterol groups.

FIG. 11. Chemical structure of telodendrimers containing eight oxalicacid groups, named as PEG^(5k)(OAOA-L-R)₄.

FIG. 12. Synthesis route for telodendrimers containing eight oxalic acidgroups, PEG^(5k)(OAOA-L-R)₄.

FIG. 13. MALDI-TOF MS of telodendrimers containing four guanidinegroups.

FIG. 14. MALDI-TOF MS of telodendrimers containing eight guanidinegroups.

FIG. 15. Hydrodynamic diameters of the BSA-loaded telodendrimerscontaining four (upper row) or eight (lower row) guanidine groups at aloading ratio of BSA to telodendrimer of 1/3 by weight in PBS (1×) at atelodendrimer concentration of 1 mg/mL after a storage at 4° C. for 2months.

FIG. 16. Hydrodynamic diameters of telodendrimers containing eight aminogroups before (upper row) and after (lower row) loading of BSA at aloading ratio of BSA to telodendrimer of 1/3 by weight in PBS (1×) at atelodendrimer concentration of 1 mg/mL.

FIG. 17. The formation of protein-polycation-telodendrimernanoparticles. The protein should be a negatively charged protein, e.g.,BSA, PEI is used as a model polycation, and a telodendrimer isPEG^(5k)(OAOA-L-R)₄.

FIG. 18. The hand-shaped chemical structures of telodendrimerscontaining four (left) or eight (right) guanidine groups.

FIG. 19. Chemical structure of crosslinkable telodendrimers.

FIG. 20. Chemical structure of the telodendrimers containing eight aminogroups, named as PEG^(5k)(LysLys-L-R)₄.

FIG. 21. Example of telodendrimer loading. (a) Loading ability oftelodendrimer nanoparticles for FITC-insulin determined by an agarosegel retention assay. (b) Loading ability of telodendrimer nanoparticlesfor GFP determined by an agarose gel retention assay. For (a) and (b)mass ratio of protein to telodendrimer is 1/3. (c) Comparison offluorescent activities of free GFP and GFP-telodendrimer nanoparticlesin PBS (1×). (d) Loading of negatively charged FITC-BSA and positivelycharged FITC-lysozyme in telodendrimer nanoparticles determined by anagarose gel retention assay. The feed mass ratio of FITC-BSA totelodendrimer is 1/3, and the feed mass ratio of FITC-lysozyme totelodendrimer is also 1/3. Telodendrimer nanoparticles cannotefficiently load FITC-lysozyme. FITC-lysozyme can form complex withFITC-BSA, and the complex can be loaded in telodendrimer nanoparticles.

FIG. 22. Loading of positively charged FITC-lysozyme and negativelycharged FITC-BSA in PEG^(5k)(OAOA-L-R)₄ telodendrimer nanoparticlesdetermined by an agarose gel retention assay. The feed mass ratio ofFITC-BSA to telodendrimer is 1/1, and the feed mass ratio ofFITC-lysozyme to telodendrimer is also 1/1. PEG^(5k)(OAOA-L-R)₄telodendrimer nanoparticles can efficiently load FITC-lysozyme.FITC-BSA-telodendrimer complexes migrated slight longer distances thanthat for free FITC-BSA, which may be contributed from the negativecharge nature of the oxalic acid groups in PEG^(5k)(OAOA-L-R)₄telodendrimers.

FIG. 23. MALDI-TOF MS of telodendrimers containing eight amino groups.

FIG. 24. Characterization of an example of a protein-polycation complex.(a) Agarose gel retention assay for FITC-BSA-PEI complex (left) andBSA-FITC-PEI complex (right) at different mass ratios of protein topolycation. (b) Hydrodymanic diameters of the BSA-PEI complexes atdifferent mass ratios of protein to polycation in PBS (1×) at a BSAconcentration of 0.2 mg/mL.

FIG. 25. Characterization of an example ofprotein/polycation/telodendrimer nanoparticles. (a) Agarose gelretention assay for FITC-BSA-PEI-Telo complex, BSA-FITC-PEI-Telocomplex, and BSA-PEI-FITC-Telo complex at different mass ratios ofprotein/polycation/telodendrimer. (b,c) Hydrodymanic diameters (b) andzeta potential (c) of BSA-PEI-Telo nanoparticles at different massratios of protein/polycation/telodendrimer in PBS (1×) at a BSAconcentration of 0.2 mg/mL. An example of a telodendrimer isPEG^(5k)(OAOA-L-CHO)₄.

FIG. 26. Loading capacity and loading efficiency of telodendrimers forFITC-BSA determined by an agarose gel retention assay. The feed massratio of protein to telodendrimer is 1/1.

FIG. 27. Cell uptake of FITC-BSA loaded in the nanoparticles made fromtelodendrimers with eight amino groups. CLSM images of HT-29 cellsincubated at 37° C. for 3 h with FITC-BSA-loaded nanoparticles ofPEG^(5k)(LysLys-L-C17)₄ (a), PEG^(5k)(LysLys-L-CHO)₄ (b), andPEG^(5k)(LysLys-L-VE)₄ (c) at a P/T ratio of 1/3. The images were takenat a magnification of 60×. The cell nuclei were stained with DAPI(blue).

FIG. 28. Hemolytic property of telodendrimers containing four (a) oreight (b) guanidine groups at different time points after the dilutedRBC suspension was mixed with telodendrimers.

DETAILED DESCRIPTION OF THE DISCLOSURE

Although claimed subject matter will be described in terms of certainembodiments and examples, other embodiments and examples, includingembodiments and examples that do not provide all of the benefits andfeatures set forth herein, are also within the scope of this disclosure.Various structural, logical, process step, and electronic changes may bemade without departing from the scope of the disclosure.

Ranges of values are disclosed herein. The ranges set out a lower limitvalue and an upper limit value. Unless otherwise stated, the rangesinclude all values to the magnitude of the smallest value (either lowerlimit value or upper limit value) and ranges between the values of thestated range.

A novel approach to design telodendrimer nanocarriers based on thestructure of a molecule of interest by the aid of computational designwas developed. Various building blocks can be introduced intotelodendrimer backbone in a precisely controlled manner. Throughcombinatorial telodendrimer synthesis, the properties of nanocarriers,e.g., size, charge and drug loading capacity/stability, can be tuned.This well-defined and highly engineerable telodendrimer platform cam beused, for example, for nanocarrier design for protein delivery.

A functionalized and spatially segregated protein nanocarrier system wasdeveloped. The nanocarrier system can be used to deliver one or moreproteins. In an embodiment, the nanocarrier system is used toencapsulate a protein by through the use of both a hydrophobic region,to fine-tune particle size, promote protein loading, and cellularuptake, and a charged hydrophilic region, for protein stabilization andloading, and improved cell-penetration properties. In an embodiment, thenanocarrier system is used to deliver one or more proteins.

In this disclosure the synthesis and engineering of a series ofwell-defined amphiphilic telodendrimers comprised of a linearpolyethylene glycol and a dendritic polyelectrolyte decorated withdifferent protein binding moieties is described. For example, theseoptimized telodendrimers can encapsulate superior amount of proteins(e.g., 30 to 200% of the telodendrimer by weight) by multivalent hybridinteractions to form stable, neutrally charged, sub-30 nm nanoparticlescapable of transporting bioactive protein across cellular membranes.This smart platform can be used, for example, for insulin delivery fordiabetes and cytotoxic protein delivery for cancer treatment.

The charged telodendrimer shown in FIG. 18 illustrates an example oftelodendrimer design that can be used, e.g., to achieve high proteinloading and cell-penetration. The various length of polyethylene glycol(ligand layer) serves as hydrophilic segments of the telodendrimer; theadjacent layer was composed of branched architecture capped withhydrophobic natural products and charged species for proteinstabilization.

The charged telodendrimers comprise multiple segments. Examples ofsegments include linear hydrophilic polymer segments, adjacent branchedfunctional segments, charged protein binding segments. Thetelodendrimers can form nanocarriers (e.g., telodendrimer micellestructures).

Definitions

As used herein, the term “protein” includes peptides (generally 50 aminoacids or less), polypeptides (generally, 100 amino acids or less), andproteins (greater than 100 amino acids). The protein can be atherapeutic protein (e.g., a cytotoxic protein or insulin). The proteincan be an antibody, enzyme, or other bioactive protein.

As used herein, the term “moiety” refers to a part (substructure) orfunctional group of a molecule that is part of the telodendrimerstructure. For example,

refers to a cholic acid moiety,

refers to a rhein moiety,

refers to a vitamin E moiety.

As used herein, the terms “dendritic polymer” or “dendritic polymermoiety” refer to branched polymers containing a focal point, a pluralityof branched monomer units, and a plurality of end groups. The monomersare linked together to form arms (or “dendritic polymer moiety”)extending from the focal point and terminating at the end groups. Thefocal point of the dendritic polymer can be attached to other segmentsof the compounds of the disclosure, and the end groups may be furtherfunctionalized with additional chemical moieties. The dendritic polymercan be composed of, for example, branched lysine and/or branchedarginine moieties.

As used herein, the term “nanocarrier” refers to a micelle resultingfrom aggregation of telodendrimer conjugates of the present disclosure.The nanocarrier has a hydrophobic core and a hydrophilic exterior.

As used herein, the terms “monomer” and “monomer unit” refer to adiamino carboxylic acid, a dihydroxy carboxylic acid, or a hydroxylamino carboxylic acid. Examples of diamino carboxylic acid groups of thepresent disclosure include, but are not limited to, 2,3-diaminopropanoic acid, 2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid(ornithine), 2,6-diaminohexanoic acid (lysine), (2-aminoethyl)-cysteine,3-amino-2-aminomethyl propanoic acid, 3-amino-2-aminomethyl-2-methylpropanoic acid, 4-amino-2-(2-aminoethyl) butyric acid and5-amino-2-(3-aminopropyl) pentanoic acid. Examples of dihydroxycarboxylic acid groups of the present disclosure include, but are notlimited to, glyceric acid, 2,4-dihydroxybutyric acid, glyceric acid,2,4-dihydroxybutyric acid, 2,2-bis(hydroxymethyl)propionic acid, and2,2-bis(hydroxymethyl)butyric acid. Examples of hydroxyl aminocarboxylic acids include, but are not limited to, serine and homoserine.One of skill in the art will appreciate that other monomer units can beused in the present disclosure. Monomers of the present disclosure canhave a bond connectivity of, for example,

For example, when a monomer is defined as a lysine moiety, with a bondconnectivity of A-Lys-B, where A and B are generic appendages, then itcan be assumed that the structure can be any one of the following:

As used herein, the term “linker” refers to a chemical moiety that links(e.g., via covalent bonds) one segment of a dendritic conjugate toanother segment of the dendritic conjugate. The types of bonds used tolink the linker to the segments of the telodendrimers include, but arenot limited to, amides, amines, esters, carbamates, ureas, thioethers,thiocarbamates, thiocarbonate, and thioureas. For example, the linker(L′, L², L³, and/or L⁴), individually at each occurrence in thetelodendrimer, can be a polyethylene glycol moiety, polyserine moiety,polyglycine moiety, poly(serine-glycine) moiety, aliphatic amino acidmoieties, 6-amino hexanoic acid moiety, 5-amino pentanoic acid moiety,4-amino butanoic acid moiety, and beta-alanine moiety. The linker canalso be a cleavable linker. In certain embodiments, combinations oflinkers can be used. For example, the linker can be an enzyme cleavablepeptide moiety, disulfide bond moiety or an acid labile moiety. One ofskill in the art will appreciate that other types of bonds can be usedin the present disclosure. In certain embodiments, the linker L′, L²,L³, and/or L⁴ can be

or a combination thereof, or other peptide sequence or spacer molecules.

As used herein, PEG group refers to polyethylene glycol. For example,the structure of PEG is

where X is selected from the group consisting of —NH₂, —OH, —SH, —COOH,—OMe, —N₃, —C═CH₂, or —≡CH, Y is selected from the group consisting of—C(═O)O—, —OC(═O)—, —OC(═O)NH—. —NHC(═O)—, —NHC(═O)O—, —NH—, —O—, —S—,

—N(PEG)-, —NHCOLys(PEG)-, —NHCO[branched Lys(PEG)]_(n)NH—, -Lys-,-Lys(PEG)-, -Lys(PEG)-Lys, -Lys(PEG)-Lys(PEG)-, Lys(PEG-Lys-Lys(PEG),and -Lys(PEG)-Lys(Lys(PEG)₂)-Lys- and n is the number of repeating unitin a range of 1 to 72736.

As used herein, the term “reversible crosslinking group” refers to achemical moiety that can be reversible reacted with another chemicalmoiety that will crosslink and decrosslink when exposed to certainconditions (e.g., different pH condition, chemical environments (e.g.sugar level), redox environments (concentration of glutathione) and UVlight of varying wavelength). For example, a coumarin derivative moiety,can be photocrosslinked at >300 nm and decrosslinked at −265 nm. Anotherexample is catechol and boronic acid which form a boronate crosslinkage,which can be cleaved at acidic pH or with cis-diol containing sugar.Another example is disulfide formation, which can be cleaved underhigher concentration of glutathione in vivo. The degree of crosslinkingcan be controlled by the density of crosslinking moieties andcrosslinking conditions, e.g., the time of reversible photocrosslinkablegroups are exposed to UV light.

As used herein, the term “oligomer” or “oligomer moiety” refers tofifteen or fewer monomers, as described above, covalently linkedtogether. The monomers may be linked together in a linear or branchedfashion. The oligomer may function as a focal point for a branchedsegment of a telodendrimer.

As used herein, the term “hydrophobic group” refers to a chemical moietythat is water-insoluble or repelled by water. Examples of hydrophobicgroups include, but are not limited to, long-chain alkanes and fattyacids, lipids, vitamins, natural compounds, herbal extracts,fluorocarbons, silicones, certain steroids such as cholesterol, bileacids, and certain polymers such as, for example, polystyrene andpolyisoprene.

As used herein, the term “hydrophilic group” refers to a chemical moietythat is water-soluble or attracted to water. Examples of hydrophilicgroups include, but are not limited to, alcohols, short-chain carboxylicacids, quaternary amines, sulfonates, phosphates, sugars, and certainpolymers such as, for example, PEG, PVA.

As used herein, the term “amphiphilic compound” refers to a compoundhaving both hydrophobic portions and hydrophilic portions. For example,the amphiphilic compounds of the present disclosure can have onehydrophilic part of the compound and one hydrophobic part of thecompound, for example, bile acids, cholic acids, riboflavin, chlorgenicacid, etc.

As used herein, the term “polar compound” refers to a compound having anon-zero vector sum of its bond dipoles.

As used herein, the terms “treat”, “treating” and “treatment” refer toany indicia of success in the treatment or amelioration of an injury,pathology, condition, or symptom (e.g., pain), including any objectiveor subjective parameter such as abatement; remission; diminishing ofsymptoms or making the symptom, injury, pathology or condition moretolerable to the patient; decreasing the frequency or duration of thesymptom or condition; or, in some situations, preventing the onset ofthe symptom or condition. The treatment or amelioration of symptoms canbe based on any objective or subjective parameter; including, e.g., theresult of a physical examination.

As used herein, the term “subject” refers to animals such as mammals.Suitable examples of mammals include, but are not limited to, primates(e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats,mice, and the like. In certain embodiments, the subject is a human.

As used herein, the terms “therapeutically effective amount or dose” or“therapeutically sufficient amount or dose” or “effective or sufficientamount or dose” refer to a dose that produces therapeutic effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms(vols. 1-3, 1992); Lloyd, The Art, Science and Technology ofPharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999);and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003,Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, thetherapeutically effective dose can often be lower than the conventionaltherapeutically effective dose for non-sensitized cells.

Charged Telodendrimers. In an aspect, the present disclosure providescharged telodendrimers. The charged telodendrimers are linear-dendriticcopolymers. The charged telodendrimers are functional segregatedtelodendrimers having, for example, two or three functional segments. Inan embodiment, the functional segments are a hydrophilic segment and ahydrophobic segment. The hydrophilic segment comprises one or morecharged groups. The charged telodendrimers may comprise an intermediatelayer. The charged telodendrimers may have one or more crosslinkinggroups (e.g., boronic acid/catechol reversible crosslinking groups).

The charged telodendrimers may comprise PEG groups that can form a PEGlayer. Without intending to be bound by any particular theory, it isconsidered that the PEG layer serves as a stealth hydrophilic shell tostabilize the nanoparticle and to avoid systemic clearance by thereticuloendothelial system (RES). The intermediate layer, if present,contains for example, optional crosslinkable functional group(s),amphiphilic oligo-cholic acid, riboflavin, or chlorogenic acid and canfurther stabilize nanoparticle and cage drug molecules in the core ofnanoparticle. The interior layer (i.e., hydrophilic layer) comprisespositively or negatively charged moieties and may comprise, for example,protein-binding building blocks, such as vitamins (e.g., α-tocopherol,riboflavin, folic acid, retinoic acid, etc.), functional lipids(ceramide), and chemical extracts (e.g., rhein, coumarin, curcurmine,etc.), from herbal medicine to increase the affinity to drug molecules.

In an embodiment, the present disclosure provides charged telodendrimersthat are functional and spatially segregated telodendrimers having 1 to128 charged groups. The telodendrimers may have one or more crosslinkinggroups (e.g., reversible boronate crosslinking groups). In anembodiment, the telodendrimers are functional segregated telodendrimershaving three functional segments.

In an embodiment the disclosure provides a compound of formula (I):

where PEG is optionally present and is a polyethylene glycol moiety,where PEG has a molecular weight of 44 Da to 100 kDa; X is optionallypresent and is a branched monomer unit; each L¹ is independentlyoptional and is a linker group; each L² is independently optional and isa linker group; each L³ is independently optional and is a linker group;each L⁴ is independently optional and is a linker group; D¹ is optionaland is a dendritic polymer moiety having one or more branched monomerunits (X), and a plurality of end groups; D² is a dendritic polymerhaving one or more branched monomer units (X), and a plurality of endgroups; R¹ is optional and is an end group of the dendritic polymer andis independently at each occurrence in the compound selected from thegroup consisting of crosslinkable groups (boronic acid, cisdiols, amine,carboxylic acids, acryl groups, epoxide, thiol groups, malaimide, C═Cdouble bond, azide, alkyne, coumarin and chlorogenic acid etc); R² is anend group of the dendritic polymer and is independently at eachoccurrence in the compound selected from the group consisting ofpositively or negatively charged groups (e.g., arginine, lysine,guanidine, amine, amidine, tetrazole, hydroxyl, carboxyl, phosphate,sulfonate, methanesulfonamide, sulfonamide, or oxalic acid functionalgroups) and neutral groups (e.g., polar groups, such as sugars,peptides, and hydrophilic polymers), or hydrophobic groups, such aslong-chain alkanes (C₁-C₅₀) and fatty acids (C₁-C₅₀), lipids, vitamins,natural compounds, herbal extracts, aromatic molecules, esters,halogens, nitrocompounds, anthracyclines, fluorocarbons, silicones,certain steroids such as cholesterol, terpenoids, vitamins, and polymers(e.g., PLGA, polycaprolactone, polylactic acid, polyglycolic acid,polystyrene and polyisoprene, polyvinyl pyridine)), or amphiphilicgroups (e.g. cholic acid, riboflavin, chlorogenic acid). The R² group(s)include at least one positively or negatively charged groups. Subscriptx is an integer from 1 to 64, where subscript x is equal to the numberof end groups on the dendritic polymer. Subscript y is an integer from 1to 64, where subscript y is equal to the number of end groups on thedendritic polymer. Subscript p is an integer from 0 to 32. Subscript mis an integer from 0 to 32.

The charged telodendrimers have one or more charged groups (e.g., R²groups). The charged groups are positively charged groups or negativelycharged groups. In an embodiment, all of the charged groups present arepositively charged groups. In an embodiment, all of the charged groupsare negatively charged groups. In an embodiment, the number of chargedgroups present in the telodendrimer is 1-128, including all integernumbers of charged groups and ranges therebetween. In an embodiment, thenumber of charged groups present in the telodendrimer is 2-64. In anembodiment, the number of charged groups present in the telodendrimer is4-16. In an embodiment, the number of charged groups present in thetelodendrimer is 4. In an embodiment, the number of charged groupspresent in the telodendrimer is 8. When D² is present and, for example,a branched arginine dendritic moiety, the guanidine portion of thearginine subunits are not part of D², but rather, the guanidine moietyis an R² group.

When X is present, in an embodiment, at each occurrence in the compound,the branched monomer unit (X) is independently selected from the groupconsisting of a diamino carboxylic acid moiety, a dihydroxy carboxylicacid moiety, and a hydroxyl amino carboxylic acid moiety.

R² is covalently bonded to a dendritic polymer or linker. The R² groupsmay be end groups. The R² groups may be linked to another R² group or R²end groups. The R² group(s) is/are independently at each occurrence inthe compound selected from the group consisting of positively ornegatively charged groups (e.g., arginine, lysine, guanidine, amine(e.g., secondary, tertiary or quaternary amines), amidine, tetrazole,hydroxyl, carboxyl, phosphate, sulfonate, methanesulfonamide,sulfonamide, or oxalic acid functional groups) and neutral groups (e.g.,polar groups: sugars, peptides, hydrophilic polymers, or hydrophobicgroups: long-chain alkanes (C1-C₅₀) and fatty acids (C1-C₅₀), lipids,vitamins, natural compounds, herbal extracts, aromatic molecules,esters, halogens, nitrocompounds, anthracyclines, fluorocarbons,silicones, certain steroids such as cholesterol, terpenoids, vitamins,and polymers (e.g., PLGA, polycaprolactone, polylactic acid,polyglycolic acid, polystyrene and polyisoprene, polyvinyl pyridine); oramphiphilic groups, cholic acid, riboflavin, chlorogenic acid) where atleast one positively or negatively charged groups are present as R²groups. R² groups may be directly bonded to the dendritic moiety (e.g.the guanidine portion of an argine moiety), or they may be attachedthrough a linker. When R² is not an end group each R² is linked to oneof the end R² groups. In an embodiment, at least one hydrophobicgroup/moiety is an R² group.

R¹, if present, is covalently bonded to a dendritic polymer or a linker.The R¹ groups may be end groups. The R¹ groups may be linked to anotherR¹ group or R¹ end groups. R¹ and can include, for example:crosslinkable groups (boronic acid, cisdiols, amine, carboxylic acids,acryl groups, epoxide, thiol groups, malaimide, C═C double bond, azide,alkyne, coumarin, and chlorogenic acid, etc.). When R¹ is not an endgroup each R¹ is linked to one of the end R¹ groups.

In various embodiments, the charged telodendrimer compound of thepresent disclosure has the following structure:

For example, each branched monomer unit is a lysine moiety or anarginine moiety or selected from a lysine moiety and an arginine moiety.

In an embodiment, at each occurrence in the compound the linker (e.g.,L¹, L², L³, and/or L⁴) are independently selected from the groupconsisting of:

In an embodiment, at each occurrence in the compound the linker (e.g.,L¹, L², L³, and/or L⁴) or a combination thereof comprises a cleavablegroup. In a specific embodiment, the cleavable group is a disulfidecleavable moiety.

In an embodiment, the PEG portion of the compound is selected from thegroup consisting of:

where each K is lysine in the compound of formula (I).

In an embodiment, 0 to 32 of R¹ groups, 1 to 32 of R² groups are chargedor neutral groups.

In an embodiment, the reversible crosslinking group (e.g., R¹), ifpresent, is a coumarin moiety, 4-methylcoumarin moiety, boronic acidmoiety or derivative or analog thereof, catechol moiety or derivative oranalog thereof, cis-diol moiety or derivative or analog thereof,cinnamic acid moiety or derivative or analog thereof, chlorogenic acidmoiety or derivative or analog thereof, amine moiety or a derivativethereof, carboxylic acid or a derivative thereof, acyl group, or aderivative thereof, epoxide or a derivative thereof, thiol group or aderivative thereof, malaimide or a derivative thereof, alkene or aderivative thereof, azide or a derivative thereof, alkyne or aderivative thereof, comarin or a derivative thereof, or a combinationthereof.

The charged group can be any group/moiety with a positive or negativecharge. For example, the charged group has a positive or negative chargein aqueous solution at a certain pH. In an embodiment, the charged group(e.g., R²) is a moiety or derivative or analog of arginine, lysine, orguanidine. In an embodiment, the charged group (e.g., R²) is an moietyor derivative or analog of an amine, amidine, tetrazole, hydroxyl,carboxyl, phosphate, sulfonate, sulfonamide (e.g., methanesulfonamide),oxalic acid, or similar functional groups.

In an embodiment, the neutral group is the moiety or derivative oranalog of sugars, peptides, hydrophilic polymers, long-chain alkanes(C₁-C₅₀) and fatty acids (C₁-C₅₀), aromatic molecules, esters, halogens,nitrocompounds, anthracyclines, fluorocarbons, silicones, certainsteroids such as cholesterol, terpenoids, vitamins, and polymers (e.g.,PLGA, polycaprolactone, polylactic acid, polyglycolic acid, polystyreneand polyisoprene, polyvinyl pyridine); amphiphilic groups, cholic acid,riboflavin, chlorogenic acid and natural compound extract and syntheticcompounds.

The charged telodendrimers can have various combinations of functionalgroups (e.g., R² and, if present R¹ groups). The functional groups canbe end groups or linked to end groups. In an embodiment, all of the R²groups present in the charged telodendrimer are all charged groups andthe R¹ groups, if present, are hydrophobic and/or crosslinking groups.In an embodiment, all of the R² groups present in the chargedtelodendrimer are charged groups or hydrophobic groups and the R¹groups, if present, are hydrophobic and/or crosslinking groups.

The dendritic moiety may comprise one or more amino acid moieties (e.g.,lysine and/or arginine moieties). For example, it is a polylysine orpolyarginine moiety. Amino acid side chains may further provideadditional branches or an R¹ or R² group (e.g., a terminal R¹ or R²group). For example, in the case of a polylysine dendritic moiety, thenitrogen of the lysine side chain may further react to form additionalbranches, or may be an R² group. Different moieties (e.g., functionalgroups) may be selectively installed at selected end groups of thedendritic moiety using orthogonal protecting group strategies.

The charged telodendrimers may be used to stabilize proteins. The typeof charge, the number of charged groups, the ratio of charged groups tohydrophobic groups (if present), the spatial orientation of the chargedgroups, and/or the density of the charged groups can be selected tostabilize a specific protein.

Nanocarriers. In an aspect, the present disclosure provides nanocarrierscomprising charged telodendrimers. Nanocarriers can also be referred toherein as nanoparticles. In an embodiment, a composition comprises anaggregate of a plurality of the telodendrimers that form a nanocarrierhaving a hydrophobic core and a hydrophilic exterior.

The nanocarrier may be a telodendrimer micelle. A telodendrimer micelleis a nanoconstruct formed by the self-assembly of the telodendrimer inaqueous solution. The telodendrimer micelle can serve as a nanocarrierto load various types of proteins.

The nanocarriers (e.g., telodendrimer micelles) have a multiple layer(e.g., a two-layer or three-layer) structure. The three-layer structurecomprises an intermediate layer.

The empty nanocarriers were examined to be nontoxic in cell culture andthe protein-loaded nanoformulations exhibited the similar potency inkilling cancer cells in vitro. The resulting nanocarriers exhibitsuperior protein loading capacity and stability. The optimizednanoparticle is able to targeted deliver the payload cytotoxic proteinsto the cancer site.

In an embodiment, the nanocarrier comprises a plurality of chargedtelodendrimer compounds. In an embodiment, the nanocarrier comprises oneor more charged proteins. The nanocarriers comprising one or morecharged proteins may have a diameter of 5 nm to 50 nm, including allinteger nm values and ranges therebetween. In an embodiment, thenanocarriers comprising one or more charged proteins may have a diameterof 10 nm to 30 nm.

The telodendrimers of the present disclosure can aggregate to formnanocarriers with a hybrid hydrophobic/polyelectrolic core, optionally,an intermediate layer (e.g., a reversible crosslinkable layer), and ahydrophilic exterior. In an embodiment, a plurality of telodendrimersaggregate to form nanocarriers with a hydrophobic and polyelectroliccore and a hydrophilic exterior. In an embodiment, the disclosureprovides a nanocarrier having an interior and an exterior, thenanocarrier comprising a plurality of the telodendrimer conjugates ofthe disclosure, wherein each compound self-assembles in an aqueoussolvent to form the nanocarrier such that a hydrophobic pocket is formedin the interior of the nanocarrier, and wherein the hydrophilic segment(e.g., PEG) of each compound self-assembles on the exterior of thenanocarrier. The telodendrimers may encapsulate or form a layer on(e.g., a layer on at least a part of) one or more protein.

The telodendrimers can be designed such that each of the proteinscarried will have a different release profile. Examples of conditionsthat can affect the release profile of carried proteins include time andbiological environment.

The nanocarrier may comprise two or more different telodendrimer/proteinconstructs. Each of the two or more different telodendrimer polymers caneach be designed for a different protein combinations (i.e., theaffinity layer of each telodendrimer can be tuned to differentproteins).

The nanocarrier can further comprise a polycation material. Examples ofpolycation materials include, but are not limited to, cationic polymerssuch as, for example, polyethylenimine (PEI), polylysine, orpoly(dimethylaminoethyl methacrylate) (PDMAEMA). Combinations ofpolycation materials can be used. In various examples, a polycationicmaterial (e.g., a polymer) has a molecular weight of 500 Daltons to 100kiloDaltons, including all integer Dalton values and rangestherebetween. Various ratios of protein(s) to polycation material(s) canbe used. For example, the ratio of protein(s) to polycation material(s)(mass ratio) is 1:1 to 1:40. In another example, the ratio of protein(s)to polycation material(s) (mass ratio) is 1:2. Various ratios ofpolycation material(s) to telodendrimer(s) can be used. For example, theratio of polycation material(s) to telodendrimer(s) (mass ratio) is1:0.05 to 1:20. In another example, the ratio of polycation material(s)to telodendrimer(s) (mass ratio) is 1:1. In an example, the ratio ofpolycation material(s) to telodendrimer(s) (mass ratio) is 1:1 to 1:40and the ratio of polycation material(s) to telodendrimer(s) (mass ratio)is 1:0.05 to 1:20.

The protein or protein mixtures can be dissolved in phosphate bufferedsaline, and telodendrimer(s) in phosphate buffered saline are rapidlyadded into protein solution. The proteins will interact (e.g., beencapsulated) mainly by the telodendrimers through electrostaticinteraction, hydrogen bonding, and hydrophobic-hydrophobic interaction.

For example, each of the telodendrimers can be associated with (e.g.,encapsulate) proteins (e.g., a different protein combinations) inseparate reactions. Subsequently, the two or more telodendrimerpolymer/protein combinations can be combined under such conditions thatthey form micelles containing a mix of telodendrimer polymer/proteinconstructs. If, for example, the micelles contain 100 or so individualtelodendrimers, it is expected that the “mixed” micelles will containstochiastic mix of the two or more proteins. The average compositionwill depend upon the ratio of the 2 or more telodendrimerpolymer/proteins constructs in the mixture. The “mixed” micelles can beused to deliver three or more proteins at the same time in apredetermined ratio (e.g., where the ratio is based on the relativestarting amounts of the 3 or more proteins).

In the “mixed” micelle embodiment, it may be desirable that eachtelodendrimer have two different end groups (R¹ and R²), where R¹ istuned for particle size, protein stability and hydrophobic interactionsand R¹ is tuned to provide a charged protein interaction andstabilization.

Some embodiments of the present disclosure provide nanocarriers whereineach amphiphilic compound R¹, le, is independently cholic acid,allocholic acid, pythocholic acid, avicholic acid, deoxycholic acid, orchenodeoxycholic acid.

Protein-loaded telodendrimer nanoparticles of the present disclosure arestable in particle sizes as detected by DLS analysis upon storage in PBSor saline at 4° C. and room temperature within the monitoring durationfor 3 months. The activity of cytotoxic protein (DTAT13) andpeptide-drug conjugates are remained the same as free therapeutics incancer cell killing in cell culture.

Protein therapeutics can be released out from the complex via thecompetition by the high concentration of serum proteins in vivo. Therelease rate can be tuned by the adjusting the protein binding affinityof telodendrimers. Therefore, protein-nanotherapeutics can beadministrated directly for in vivo use without the need to purificationof the released protein.

The telodendrimers of the present disclosure can be used to, forexample, encapsulate antibodies and other therapeutic proteins andincrease the therapeutic index mainly in twofold: (1) to improve thestability of the protein therapeutics, e.g., antitoxin antibodies,during storage even at room temperature for the possible applications atrural area and military use. They can be developed as onsite-careformulations for direct administration. Antibodies will be released uponserum albumin and IgG competition of nanocarrier. It is also useful tostabilize antibody drugs for routine use to prevent the denaturation dueto aggregation. (2) An even broader application is to deliver proteinand antibodies reagents into cells, for example, antibodies againstintracellular proteins used in biochemistry assays or pathologydetections, therefore becoming therapeutic to treat various diseases.

The charged telodendrimers can be present in a composition. In anembodiment, the composition comprises one or more chargedtelodendrimers. The composition may comprise a mixture of positivelycharged telodendrimers, a mixture of negatively charged telodendrimers,or a mixture of positively and negatively charged telodendrimers. In anembodiment the composition further comprises one or more proteins. In anembodiment the composition further comprises one or more drugs. Thecomposition can have a formulation as disclosed herein. For example, thecomposition can be a pharmaceutical composition as described herein.

Any charged protein can be used. For example, the protein is apositively charged or negatively charged protein. In an embodiment, theprotein is an imaging agent-labeled protein. In an embodiment, thecomposition comprises or nanocarriers encapsulate an amount ofprotein(s) that is 30-200% of the telodendrimer present in thecomposition or nanocarriers by weight, including all integer weight %values and ranges therebetween.

Examples of therapeutic proteins that can be used includenucleoproteins, glycoproteins, lipoproteins, immunotherapeutic proteins,porcine somatotropin for increasing feed conversion efficiency in a pig,insulin, growth hormone, buserelin, leuprolide, interferon, gonadorelin,calcitonin, cyclosporin, lisinopril, captopril, delapril, tissueplasminogen activator, epidermal growth factor, fibroblast growth factor(acidic or basic), platelet derived growth factor, transforming growthfactor (alpha or beta), vasoactive intestinal peptide, tumor necrosisfactor; hormones such as glucagon, calcitronin, adrecosticotrophichormone, follicle stimulating hormone, enkaphalins, β-endorphin,somatostin, gonado trophine, α-melanocyte stimulating hormone.Additional examples include bombesin, atrial naturiuretic peptides andluteinizing hormone releasing (LHRH), substance P, vasopressins,α-globulins, transferrins, fibrinogens, β-globulins, prothrombin(bovine), ceruloplasmin, a₂-glycoproteins, a₂-globulins, fetuin(bovine), albumin and prealbumin, bovine serum albumin, greenfluorescent protein, diphtheria toxins, lysozyme, trypsin, cytochrome c,saporin, ribonuclease A, IgG, and antibodies.

The nanocarriers may comprise one or more drugs. The drugs can betherapeutic agents. The drugs may be sequestered in the nanocarriers(e.g., sequestered in one or more of the layers of a telodendrimer) orlinked to the conjugates of the present disclosure. Examples of drugsinclude, but are not limited to, cytostatic agents, cytotoxic agents(such as for example, but not limited to, DNA interactive agents (suchas cisplatin or doxorubicin)); taxanes (e.g., taxotere, taxol);topoisomerase II inhibitors (such as etoposide); topoisomerase Iinhibitors (such as irinotecan (or CPT-11), camptostar, or topotecan);tubulin interacting agents (such as paclitaxel, docetaxel or theepothilones); hormonal agents (such as tamoxifen); thymidilate synthaseinhibitors (such as 5-fluorouracil); anti-metabolites (such asmethotrexate); alkylating agents (such as temozolomide (TEMODAR™ fromSchering-Plough Corporation, Kenilworth, N.J.), cyclophosphamide);aromatase combinations; ara-C, adriamycin, cytoxan, and gemcitabine.Other drugs useful in the nanocarrier of the present disclosure includebut are not limited to Uracil mustard, Chlormethine, Ifosfamide,Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, oxaliplatin, leucovirin,oxaliplatin (ELOXATIN™ from Sanofi-Synthelabo Pharmaceuticals, France),Pentostatine, Vinblastine, Vincristine, Vindesine, Bleomycin,Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin,Mithramycin, Deoxycoformycin, Mitomycin-C, L-Asparaginase, Teniposide17alpha-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone,Fluoxymesterone, Dromostanolone propionate, Testolactone,Megestrolacetate, Methylprednisolone, Methyltestosterone, Prednisolone,Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide,Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide,Toremifene, goserelin, Cisplatin, Carboplatin, Hydroxyurea, Amsacrine,Procarbazine, Mitotane, Mitoxantrone, Levamisole, Navelbene,Anastrazole, Letrazole, Capecitabine, Reloxafine, Droloxafine, orHexamethylmelamine. Prodrug forms are also useful in the disclosure.

In an aspect, the present disclosure provides methods of using thetelodendrimers. The telodendrimers can be used, for example, in methodsof treatment.

Method of treating. The compositions or nanocarriers of the presentdisclosure can be used to treat any disease requiring the administrationof a protein, such as, for example, by sequestering a protein in theinterior of the nanocarrier, and delivering said protein to a target.The protein(s) can be delivered systemically or intracellularly. In anembodiment, compositions comprising the telodendrimers are used in amethod for treating a disease.

In some embodiments, the present disclosure provides a method oftreating a disease, including administering to a subject in need of suchtreatment a therapeutically effective amount of a composition ornanocarrier of the present disclosure, where the nanocarrier includes anencapsulated protein.

The compositions or nanocarriers of the present disclosure can beadministered to a subject for treatment, e.g., of hyperproliferativedisorders including cancer such as, but not limited to: carcinomas,gliomas, mesotheliomas, melanomas, lymphomas, leukemias,adenocarcinomas, breast cancer, ovarian cancer, cervical cancer,glioblastoma, leukemia, lymphoma, prostate cancer, and Burkitt'slymphoma, head and neck cancer, colon cancer, colorectal cancer,non-small cell lung cancer, small cell lung cancer, cancer of theesophagus, stomach cancer, pancreatic cancer, hepatobiliary cancer,cancer of the gallbladder, cancer of the small intestine, rectal cancer,kidney cancer, bladder cancer, prostate cancer, penile cancer, urethralcancer, testicular cancer, cervical cancer, vaginal cancer, uterinecancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenalcancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skincancer, retinoblastomas, multiple myelomas, Hodgkin's lymphoma, andnon-Hodgkin's lymphoma (see, CANCER: PRINCIPLES AND PRACTICE (DeVita, V.T. et al. eds 2008) for additional cancers).

Other diseases that can be treated by the compositions or nanocarriersof the present disclosure include: (1) inflammatory or allergic diseasessuch as systemic anaphylaxis or hypersensitivity responses, drugallergies, insect sting allergies; inflammatory bowel diseases, such asCrohn's disease, ulcerative colitis, ileitis and enteritis; vaginitis;psoriasis and inflammatory dermatoses such as dermatitis, eczema, atopicdermatitis, allergic contact dermatitis, urticaria; vasculitis;spondyloarthropathies; scleroderma; respiratory allergic diseases suchas asthma, allergic rhinitis, hypersensitivity lung diseases, and thelike, (2) autoimmune diseases, such as arthritis (rheumatoid andpsoriatic), osteoarthritis, multiple sclerosis, systemic lupuserythematosus, diabetes mellitus, glomerulonephritis, and the like, (3)graft rejection (including allograft rejection and graft-v-hostdisease), and (4) other diseases in which undesired inflammatoryresponses are to be inhibited (e.g., atherosclerosis, myositis,neurological conditions such as stroke and closed-head injuries,neurodegenerative diseases, Alzheimer's disease, encephalitis,meningitis, osteoporosis, gout, hepatitis, nephritis, sepsis,sarcoidosis, conjunctivitis, otitis, chronic obstructive pulmonarydisease, sinusitis and Behcet's syndrome).

In addition, the compositions or nanocarriers of the present disclosureare useful for the treatment of infection by pathogens such as viruses,bacteria, fungi, and parasites. Other diseases can be treated using thecompositions or nanocarriers of the present disclosure.

Formulations. The nanocarriers of the present disclosure can beformulated in a variety of different manners known to one of skill inthe art. Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereare a wide variety of suitable formulations of pharmaceuticalcompositions of the present disclosure (see, e.g., Remington'sPharmaceutical Sciences, 20^(th) ed., 2003, supra). Effectiveformulations include oral and nasal formulations, formulations forparenteral administration, and compositions formulated for with extendedrelease.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of a compound of the presentdisclosure suspended in diluents, such as water, saline or PEG 400; (b)capsules, sachets, depots or tablets, each containing a predeterminedamount of the active ingredient, as liquids, solids, granules orgelatin; (c) suspensions in an appropriate liquid; (d) suitableemulsions; and (e) patches. The liquid solutions described above can besterile solutions. The pharmaceutical forms can include one or more oflactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch,potato starch, microcrystalline cellulose, gelatin, colloidal silicondioxide, talc, magnesium stearate, stearic acid, and other excipients,colorants, fillers, binders, diluents, buffering agents, moisteningagents, preservatives, flavoring agents, dyes, disintegrating agents,and pharmaceutically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, e.g., sucrose, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin or sucrose and acacia emulsions, gels, and the like containing,in addition to the active ingredient, carriers known in the art.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form. The composition can, if desired, also contain othercompatible therapeutic agents. Preferred pharmaceutical preparations candeliver the compounds of the disclosure in a sustained releaseformulation.

Pharmaceutical preparations useful in the present disclosure alsoinclude extended-release formulations. In some embodiments,extended-release formulations useful in the present disclosure aredescribed in U.S. Pat. No. 6,699,508, which can be prepared according toU.S. Pat. No. 7,125,567, both patents are incorporated herein byreference.

The pharmaceutical preparations are typically delivered to a mammal,including humans and non-human mammals. Non-human mammals treated usingthe present methods include domesticated animals (e.g., canine, feline,murine, rodentia, and lagomorpha) and agricultural animals (e.g.,bovine, equine, ovine, porcine).

In practicing the methods of the present disclosure, the pharmaceuticalcompositions can be used alone, or in combination with other therapeuticor diagnostic agents.

Administration. The nanocarriers of the present disclosure can beadministered as frequently as necessary, including hourly, daily, weeklyor monthly. The compounds utilized in the pharmaceutical method of thedisclosure are administered at the initial dosage of about 0.0001 mg/kgto about 1000 mg/kg daily. A daily dose range of about 0.01 mg/kg toabout 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or about 1 mg/kgto about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg, can be used.The dosages, however, may be varied depending upon the requirements ofthe patient, the severity of the condition being treated, and thecompound being employed. For example, dosages can be empiricallydetermined considering the type and stage of disease diagnosed in aparticular patient. The dose administered to a patient, in the contextof the present disclosure should be sufficient to effect a beneficialtherapeutic response in the patient over time. The size of the dose alsowill be determined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular compoundin a particular patient. Determination of the proper dosage for aparticular situation is within the skill of the practitioner. Generally,treatment is initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached. For convenience, the total daily dosage may be divided andadministered in portions during the day, if desired. Doses can be givendaily, or on alternate days, as determined by the treating physician.Doses can also be given on a regular or continuous basis over longerperiods of time (weeks, months or years), such as through the use of asubdermal capsule, sachet or depot, or via a patch or pump.

The pharmaceutical compositions can be administered to the patient in avariety of ways, including topically, parenterally, intravenously,intradermally, subcutaneously, intramuscularly, colonically, rectally orintraperitoneally. Preferably, the pharmaceutical compositions areadministered parenterally, topically, intravenously, intramuscularly,subcutaneously, orally, or nasally, such as via inhalation.

In practicing the methods of the present disclosure, the pharmaceuticalcompositions can be used alone, or in combination with other therapeuticor diagnostic agents. The additional proteins used in the combinationprotocols of the present disclosure can be administered separately orone or more of the proteins used in the combination protocols can beadministered together, such as in an admixture. Where one or moreproteins are administered separately, the timing and schedule ofadministration of each protein can vary.

Method of imaging. In an aspect, compositions or nanocarriers comprisingcharged telodendrimers are used in imaging methods. In an embodiment, acomposition or nanocarrier comprises an imaging agent.

In an embodiment, the present disclosure provides a method of imaging,including administering to a subject to be imaged, an effective amountof a composition or nanocarrier of the present disclosure, wherein thecomposition or nanocarrier includes an imaging agent. In otherembodiments, the method of treating and the method of imaging areaccomplished simultaneously using a nanocarrier having a therapeuticprotein, and/or an imaging agent-labeled protein.

Exemplary imaging agents include paramagnetic agents, optical probes,and radionuclides. Paramagnetic agents imaging agents that are magneticunder an externally applied field. Examples of paramagnetic agentsinclude, but are not limited to, iron particles including nanoparticles.Optical probes are fluorescent compounds that can be detected byexcitation at one wavelength of radiation and detection at a second,different, wavelength of radiation. Optical probes useful in the presentdisclosure include, but are not limited to, Cy5.5, Alexa 680, Cy5, DiD(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate)and DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanineiodide). Other optical probes include quantum dots. Radionuclides areelements that undergo radioactive decay. Radionuclides useful in thepresent disclosure include, but are not limited to, ³H, ¹¹C, ¹³N, ¹⁸F,¹⁹F, ⁶⁰Co, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁸²Rb, ⁹⁰Sr, ⁹⁰Y, ⁹⁹Tc, ^(99m)Tc, ¹²³I,¹²⁴I, ¹²⁵I, ¹²⁹I, ¹³¹I, ¹³⁷Cs, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹A, Rn, Ra, Th, UPu, and ²⁴¹Am.

The steps of the method described in the various embodiments andexamples disclosed herein are sufficient to carry out the methods of thepresent disclosure. Thus, in an example, the method consists essentiallyof a combination of the steps of the methods disclosed herein. Inanother example, the method consists of such steps.

The following Statements describe various examples of the polymers andmethods of the present disclosure:

Statement 1. A compound of formula (I):

where PEG is optionally present and is a polyethylene glycol moiety,where PEG has a molecular weight of 44 Da to 100 kDa; X is optionallypresent and is a branched monomer unit; each L¹ is independentlyoptional and is a linker group; each L² is independently optional and isa linker group; each L³ is independently optional and is a linker group;each L⁴ is independently optional and is a linker group; D¹ is optionaland is a dendritic polymer moiety having one or more branched monomerunits (X), and a plurality of end groups; D² is a dendritic polymerhaving one or more branched monomer units (X), and a plurality of endgroups; R¹ is optional and is an end group of the dendritic polymer andis independently at each occurrence in the compound selected from thegroup consisting of crosslinkable groups (boronic acid, cis diols,amine, carboxylic acids, acryl groups, epoxide, thiol groups, malaimide,C═C double bond, azide, alkyne, coumarin and chlorogenic acid etc); R²is an end group of the dendritic polymer and is independently at eachoccurrence in the compound selected from the group consisting ofpositively or negatively charged groups (e.g., arginine, lysine,guanidine, amine, amidine, tetrazole, hydroxyl, carboxyl, phosphate,sulfonate, methanesulfonamide, sulfonamide, or oxalic acid functionalgroups) and neutral groups (e.g., polar groups: sugars, peptides,hydrophilic polymers, or hydrophobic groups: long-chain alkanes (C₁-C₅₀)and fatty acids (C₁-C₅₀), aromatic molecules, esters, halogens,nitrocompounds, anthracyclines, fluorocarbons, silicones, certainsteroids such as cholesterol, terpenoids, vitamins, and polymers (e.g.,PLGA, polycaprolactone, polylactic acid, polyglycolic acid, polystyreneand polyisoprene, polyvinyl pyridine), and amphiphilic groups, cholicacid, riboflavin, chlorogenic acid), where at least one positively ornegatively charged groups are present in R²; subscript x is an integerfrom 1 to 64, where subscript x is equal to the number of end groups onthe dendritic polymer; subscript y is an integer from 1 to 64, wheresubscript y is equal to the number of end groups on the dendriticpolymer; subscript p is an integer from 0 to 32; and subscript m is aninteger from 0 to 32.Statement 2. A compound according to Statement 1, where at eachoccurrence in the compound the branched monomer unit (X) isindependently selected from the group consisting of a diamino carboxylicacid moiety, a dihydroxy carboxylic acid moiety, and a hydroxyl aminocarboxylic acid moiety.Statement 3. A compound according to Statement 2, where at eachoccurrence in the compound the diamino carboxylic acid is independentlyselected from the group consisting of 2,3-diamino propanoic acid,2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid (ornithine),2,6-diaminohexanoic acid (lysine), (2-Aminoethyl)-cysteine,3-amino-2-aminomethyl propanoic acid, 3-amino-2-aminomethyl-2-methylpropanoic acid, 4-amino-2-(2-aminoethyl) butyric acid, and5-amino-2-β-aminopropyl) pentanoic acid.Statement 4. A compound according Statement 2, where the diaminocarboxylic acid moiety is an amino acid moiety.Statement 5. A compound according to any one of the precedingStatements, where each branched monomer unit X is lysine moiety.Statement 6. A compound according to any one of the precedingStatements, where the compound is selected from the group consisting of:

where each branched monomer unit is individually selected from a lysinemoiety (e.g., a polylysine moiety) and arginine (e.g., polyarginine)moiety.Statement 7. A compound according to any one of the precedingStatements, where at each occurrence in the compound the linker L¹, L²,L³ and L⁴ each are independently selected from the group consisting of apolyethylene glycol moiety, polyserine moiety, enzyme cleavable peptidemoiety, disulfide bond moiety and acid labile moiety, polyglycinemoiety, poly(serine-glycine) moiety, aliphatic amino acid moieties,6-amino hexanoic acid moiety, 5-amino pentanoic acid moiety, 4-aminobutanoic acid moiety, and beta-alanine moiety.Statement 8. A compound according to any one of the precedingStatements, where at each occurrence in the compound the linker L¹, L²,L³ and L⁴ are independently selected from the group consisting of:

Statement 9. A compound according to any one of the precedingStatements, wherein one or more linker (L¹, L², L³, L⁴ or a combinationthereof) comprises a cleavable group.Statement 10. A compound according to Statement 9, where the cleavablegroup is a disulfide cleavable moiety.Statement 11. A compound according to any one of the precedingStatements, where the (PEG)_(m) portion of the compound is selected fromthe group consisting of:

where each K is lysine.Statement 12. A compound according to any one of the precedingStatements, where at least one (e.g., 1 to 128) of the R² groups arecharged groups and, optionally, at least one of the R² groups areneutral groups, and, optionally, at least one of (e.g., 1 to 128) of theR¹ groups, if present, are reversible crosslinking groups.Statement 13. A compound according to Statement 12, where the reversiblecrosslinking group(s) is/are coumarin moiety, 4-methylcoumarin moiety,boronic acid moiety or derivative or analog thereof, catechol moiety orderivative or analog thereof, cis-diol moiety or derivative or analogthereof, cinnamic acid moiety or derivative or analog thereof,chlorogenic acid moiety or derivative or analog thereof, amine moiety ora derivative thereof, carboxylic acid or a derivative thereof, acylgroup, or a derivative thereof, epoxide or a derivative thereof, thiolgroup or a derivative thereof, malaimide or a derivative thereof, alkeneor a derivative thereof, azide or a derivative thereof, alkyne or aderivative thereof, coumarin or a derivative thereof, or a combinationthereof.Statement 14. A compound according to Statement 12, where the chargedgroup is the moiety or derivative or analog of arginine, lysine,guanidine, amine, amidine, tetrazole, hydroxyl, carboxyl, phosphate,sulfonate, methanesulfonamide, sulfonamide, or oxalic acid andfunctional groups Statement 15. A compound according to any one of thepreceding Statement 12, where the neutral group is the moiety orderivative or analog of sugars, peptides, hydrophilic polymers,long-chain alkanes (C₁-C₅₀) and fatty acids (C₁-C₅₀), aromaticmolecules, esters, halogens, nitrocompounds, anthracyclines,fluorocarbons, silicones, certain steroids such as cholesterol,terpenoids, vitamins, and polymers (e.g., PLGA, polycaprolactone,polylactic acid, polyglycolic acid, polystyrene and polyisoprene,polyvinyl pyridine); amphiphilic groups, cholic acid, riboflavin,chlorogenic acid and natural compound extract and synthetic compounds.Statement 16. A nanocarrier comprising a plurality of compoundsaccording to any one of the preceding Statements.Statement 17. A nanocarrier according to Statement 16, where thenanocarrier further comprises one or more charged proteins.Statement 18. A nanocarrier according to any one of Statements 16 or 17,where the nanocarrier further comprises one or more polycation material(e.g., one or more cationic polymer).

The following example is presented to illustrate the present disclosure.It is not intended to limiting in any manner.

Example 1

The following is an example of the preparation, characterization, anduse of charged telodendrimers of the present disclosure.

The interplay of hydrogen bonding, electrostatic and hydrophobicinteractions stabilizes and maintains protein three-dimensionalstructures. Polar amino acids, including both positive and negativecharged ones, are mostly displayed on the surface of protein to maintaindispersion in aqueous solution. In addition, the hydrophobic residuesaggregate into the hydrophobic groves to minimize solvent exposure. Thedesign of polymers to target both hydrophobic grove and polar groups onthe protein surface represents a promising solution for protein coating.The multivalent interactions can significantly increase the bindingaffinity between telodendrimer and proteins. The combination ofelectrostatic and hydrophobic moieties in telodendrimer is important forprotein binding kinetically and thermodynamically. Inspired by thecooperativity and/or multivalency effects in biological systems, wedeveloped a hand-shaped hybrid telodendrimer of a linear polyethyleneglycol (PEG) and a dendritic polyelectrolyte decorated with differenthydrophobic natural compounds tethered by a flexible linker. Wehypothesized the design of telodendrimers with flexible and dendritichybrid multiple functional groups will match protein surface curvaturesby both electrostatic and hydrophobic interactions, which serveapproaching and annealing functions, respectively, to stabilize thenanoconstructs. These multivalent hybrid interactions ensure the stablesingle layer protein-coating by telodendrimers in aqueous solution,thereby yielding nanoparticles of 10-30 nm in size capable of stablyloading high amount of proteins. To this end, four or eight guanidinegroups were introduced in the dendritic polyamino acids of thetelodendrimers to optimize the protein loading and cell-penetrationproperties, and diverse natural compounds including heptadecanoic acid(C17), cholesterol (CHO) and vitamin E (VE, D-α-tocopherol) wereselected as hydrophobic moieties in the telodendrimer nanoparticles tofine-tune the particle size and to further promote the protein loadingand cellular uptake. The resultant protein-loaded telodendrimernanoparticles of <30 nm in diameter have neutral zeta potential (<±5 mV)and high protein loading capacities (>30% of the nanoparticles byweight), and they are colloidally stable for months in phosphatebuffered saline (PBS). The telodendrimer nanoparticles can efficientlydeliver proteins such as cytotoxins intracellularly to cancer cellswhile maintaining protein bioactivity, leading to desired cell death.

Herein we report the synthesis and engineering of a series ofwell-defined amphiphilic telodendrimers comprised of a linearpolyethylene glycol and a dendritic polyelectrolyte decorated withdifferent hydrophobic natural compounds. The structure optimizationstudies showed that both charge interactions and hydrophobicinteractions were essential for protein coating/encapsulation. Further,the nature of the charged group and the structure of hydrophobicsegments can be optimized for efficient and stable proteinencapsulation. These optimized telodendrimers can encapsulate superioramount of proteins (30-200% of the telodendrimer by weight) to formneutral and stable, sub-30 nm nanoparticles capable of deliveringbioactive protein across cellular membranes. Such highly engineerabletelodendrimers allows for the fine tune of the density, location andionic strength of the charge groups, as well as the tether length andthe structure of the hydrophobic segments to design nanocarriers basedon the protein structures, e.g., charge density, structure of thehydrophobic groves. The reversibly crosslinkable functional groups couldbe introduced in the telodendrimers to further stabilize proteinencapsulation, which is responsive to biological/pathologicalenvironments, e.g., glucose level in blood or acidic tumor extracellularor lysosome pH for telodendrimer decrosslinking to release proteintherapeutics on demand to treat disease efficiently. This smart platformhas been designed specifically for insulin delivery for diabetes andcytotoxic protein delivery for cancer treatment, respectively.

Reversible crosslinking groups can be introduced in the adjacent layerof the teloenderimer to enable nanoparticle crosslinking after proteinloading. The reversible crosslinkages, e.g. boronate ester and acidlabile acylhydrazone, redox sensitive S—S bonds can respond to thebiological or pathological environment to release the protein on demandto achieve better specificity and efficacy.

Synthesis of telodendrimer. We would make the above mentionedtelodendimer via step-wise peptide chemistry. Briefly, the telodendimerwould be synthesized using a solution-phase condensation reactionstarting from MeO-PEG-NH₂.HCl. Orthogonally protected peptides such as(Fmoc)Lys(Fmoc)-OH, (Fmoc)Lys(Boc)-OH, and Fmoc-Arg(Pbf)-OH are reactedwith the N terminus of PEGylated molecules using diisopropyl carbodimideand N-hydroxybenzotriazole as coupling reagents until a negative Kaisertest result is obtained, indicating completion of the coupling reaction.PEGylated molecules are precipitated through the addition of the coldether and then washed with cold ether twice. Fmoc groups are removed bythe treatment with 4-methylpiperidine in dimethylformamide, and Boc andPbf groups are removed via the treatment with trifluoroacetic acid indichloromethane. The linker molecules and hydrophobic molecules arecoupled to the de-protected amino groups of the PEGylated molecules toyield the telodendrimer, wherein the positively charged groups areobtained upon the de-protection of Fmoc, Boc or Pbf on the orthogonallyprotected peptides.

For introducing other charged moieties instead of side chains of lysineand arginines, free amino groups on lysine side chain will be used toconjugate positively or negatively charged groups, e.g. secondary,tertiary and quaternary amines or negative carboxylic, oxalic, sulfonicor phohsphyrilic acids, etc.

Results and Discussion. Synthesis and Characterization ofTelodendrimers. The design of the telodendrimer with both guanidinegroups and hydrophobic compounds lies in follow: The positively chargedguanidine groups in the telodendrimers are supposed to interact withoppositely charged groups on protein surface while theflexible-linker-conjugated hydrophobic groups are expected to bind thehydrophobic residues of proteins to offer dual supramolecularinteractions to enforce the binding to proteins. The telodendrimers weresynthesized via step-wise peptide chemistry. The telodendrimerscontaining four guanidine groups and four hydrophobic molecules such asC17, CHO and VE are noted as PEG^(5k)(Arg-L-C17)₄, PEG^(5k)(Arg-L-CHO)₄and PEG^(5k)(Arg-L-VE)₄, respectively, and the eight guanidinegroup-containing ones are named as PEG^(5k)(ArgArg-L-C17)₄,PEG^(5k)(ArgArg-L-CHO)₄ and PEG^(5k)(ArgArg-L-VE)₄. Their chemicalstructures are displayed in FIG. 18, and their synthesis routes areshown in Schemes 8 and 9. The telodendrimers were characterized byMALDI-TOF mass spectrometry (MS) and proton nuclear magnetic resonance(NMR), and the results are displayed in Table 1 and FIGS. 13 and 14.Table 1 shows that the molecular weights of the telodendrimersdetermined by MALDI-TOF MS are very close to the theoretical values. The41 NMR spectra for the telodendrimers in DMSO-d₆ also confirm theirwell-defined chemical structures. The telodendrimers can self-assembleinto micelles in PBS due to microphase segregation, yieldingmonodispersed nanoparticles with hydrodynamic diameters (D_(h)) of 11-32nm and neutral zeta potential (Table 1). The morphology of thetelodendrimer nanoparticles was characterized by transmission electronmicroscopy (TEM). The telodendrimers containing C17 or CHO as theperipheral groups formed spherical micelles while the VE-containingtelodendrimers tend to produce heterogeneous nanoparticles that arespherical and cylindrical in shape due to the pi-pi stacking between VEgroups. The particle sizes measured from the TEM images acquire a goodagreement with the DLS results. The critical micelle concentrations(CMCs) of the telodendrimers in PBS are in a range from 1.14 to 2.68which were determined by a fluorescent method employing Nile red as aprobe (Table 1. The low CMCs of the telodendrimers suggest the formationof stable micelles in a wide concentration range.

Incorporation of Proteins within Telodendrimer Nanoparticles. Negativelycharged proteins can be effectively loaded in the telodendrimernanoparticles mainly based on electrostatic and hydrophobicinteractions. An agarose gel retention assay was used tosemi-quantitatively determine the loading capacity of the telodendrimernanoparticles for proteins, where the free proteins and protein-loadedtelodendrimer nanoparticles could be separated based on theirdifferences in size and charge. Fluorescein isothiocyanate (FITC)labeled bovine serum albumin (BSA), noted as FITC-BSA, was used as afluorescent model protein for probing the protein position in theagarose gel. A constant amount of FITC-BSA without and withtelodendrimer nanoparticles at a protein to telodendrimer (P/T) massratio of 2/1 (FIG. 2) were loaded in the agarose gel (1.5% wt). After a2 h of running in Tris-acetate-EDTA (TAE) buffer, FITC-BSA withouttelodendrimers migrated a certain distance towards the anode, while theFITC-BSA were trapped in the wells when they were loaded in thetelodendrimer nanoparticles due to the large sizes and neutral surfacecharges of the protein-loaded nanoparticles. Excess FITC-BSA in theprotein-telodendrimer systems also migrated a distance roughly equal tothat for FITC-BSA without telodendrimers. The loading capacities of thetelodendrimer nanoparticles for FITC-BSA were calculated from thefluorescence signals of the bands for unloaded FITC-BSA. As shown inFIG. 2, all the telodendrimer nanoparticles can efficiently incorporateFITC-BSA with loading capacities of more than 30% by weight. The loadingcapacity and loading efficiency for the telodendrimers containing eightguanidine groups are higher than that for the telodendrimers containingfour guanidine groups, and C17-containing telodendrimers have higherprotein loading capacities when compared to the CHO- or VE-containingones. Among these telodendrimers, PEG^(5k)(ArgArg-L-C17)₄ highlighted bya dotted square in FIG. 2 shows the best protein loading behavior with aloading capacity of 200% and a loading efficiency of 100% for FITC-BSA.These facts indicate that the precise control on the numbers ofguanidine groups and the species of hydrophobic groups is critical tooptimize the protein loading behaviors of the telodendrimernanoparticles.

The binding affinities of proteins to telodendrimers were measured byisothermal titration calorimetry (ITC). BSA and the telodendrimerscontaining eight guanidine groups with different hydrophobic moietieswere dissolved in PBS (1×), and BSA was titrated into the telodendrimersolutions at 37° C. As shown in FIG. 3, the equilibria for thetelodendrimers containing rigid CHO or VE hydrophobic molecules wereexothermic and defined the overall negative enthalpy of the complexationprocess (FIGS. 3b and 3c ). The individual peaks in the thermograms wereintegrated by the instrument software and the isotherms were fittedusing one-site binding model to yield the stoichiometry, bindingaffinity (K_(a)), binding enthalpy (ΔH), which are displayed in Table 2.The numbers of BSA equivalents incorporated into telodendrimernanoparticles are in a range of 0.09 to 0.15, which means the P/T massratios in the protein-telodendrimer nanoparticles are close to 1/1,confirming that high amounts of proteins can bind to the telodendrimers.The binding affinities of CHO- and VE-containing telodendrimers for BSAare 1.8×10⁵ and 3.5×10⁵, respectively (Table 2).

To further confirm the binding between telodendrimer and protein,FITC-labeled PEG^(5k)(ArgArg-L-C17)₄ (noted asFITC-PEG^(5k)(ArgArg-L-C17)₄) and Rhodamine B-labeled BSA (noted asRB-BSA) molecules were selected as a donor-acceptor pair to investigatethe molecular proximity by Förster resonance energy transfer (FRET)technique. The data shows that FITC-PEG^(5k)(ArgArg-L-C17)₄ could beexcited by a light source with a wavelength of 439 nm, and it showed aemission peak centered at 528 nm, while RB-BSA could hardly be excited.When using the same light source to excite 1/1 (w/w) mixture ofFITC-PEG^(5k)(ArgArg-L-C17)₄ and RB-BSA, except the peak forFITC-PEG^(5k)(ArgArg-L-C17)₄ at 528 nm, another peak centered at 584 nmappeared, which related to RB-BSA. It testified the FRET of thisdonor-acceptor pair that occurred when the distance between the proteinand telodendrimer was less than 10 nm, indicating the strongprotein-telodendrimer binding. In comparison, no obvious FRET wasobserved for the mixture of RB-BSA and FITC-BSA, while the addition ofPEG^(5k)(ArgArg-L-C17)₄ to mixture of RB-BSA and FITC-BSA to reach a P/Tratio of 1/1 by weight led to a significant increase in fluorescence at584 nm and decrease in fluorescence at 528 nm, that was also observed inthe protein-telodendrimer systems with other telodendrimer species orP/T mass ratios. This fact indicates that complexation withtelodendrimers brings the proteins with two different dyes together. Wesuggest that the RB-BSA loaded in the telodendrimer nanoparticles can bereleased upon the disintegration of the supramolecular structures and ina manner of exchange within other proteins in serum, such as BSA. Toclarify it, different amounts of BSA were added in the mixtures ofFITC-PEG^(5k)(ArgArg-L-C17)₄ and RB-BSA (1/1, w/w), followed by a 4 hincubation at room temperature. We found that the normalized FRET ratio(which was calculated by the formula of [100%×I₅₈₄/(I₅₈₄+I₅₂₈)], whereI₅₈₄ and I₅₂₈ were fluorescence intensities of RB-BSA at 584 nm andFITC-PEG^(5k)(ArgArg-L-C17)₄ at 528 nm) generally decreased withincreasing BSA concentration (FIG. 4c ), indicating dissociation of thetelodendrimer nanoparticles with the payload protein in the presence ofhigh amounts of free BSA. This suggested the possible pathway for thepayload proteins to be released from the telodendrimer nanoparticlesthrough exchange with other proteins, which was also affirmed by anagarose gel retention assay.

The kinetics of protein-telodendrimer interactions were measured bybio-layer interferometry (BLI). Streptavidin biosensors were prewettedin BSA solution since BSA would occupy most of the nonspecific bindingsites on the sensor surfaces, and the association was carried out intelodendrimer solution followed by dissociation in BSA solutions withdifferent concentrations at 37° C. (FIG. 4a ). As shown in FIG. 4b ,diverse dissociation behaviors are found in different dissociationbuffers after binding of PEG^(5k)(ArgArg-L-C17)₄ telodendrimers. Thedissociation is not efficient in PBS indicating the strong bindingbetween telodendrimers and proteins. The existence of BSA indissociation buffers significantly accelerates telodendrimerdissociation. Moreover, the dissociation rate constant (k_(off))increases with increasing BSA concentration in dissociation buffers(FIG. 4c ), which acquires reasonable agreement with the proteinexchange mechanism for protein release from telodendrimer nanoparticlesdemonstrated from the FRET results. It is a general rule that thebinding responses should contain decays in the signal of at least 5%during the dissociation phase of the binding cycle to define a k_(off).To achieve efficient dissociation and mimic serum concentration in vivo,40 mg/mL of BSA solution is selected as the dissociation buffer forfollowing BLI studies. Multiple concentrations of telodendrimers wereused in the association step for different sensors in order to fitresults globally and accurately get the binding affinities oftelodendrimers to proteins. PEG^(5k)(ArgArg-L-C17)₄ is taken as anexample to demonstrate global analysis of the BLI data, and the kineticsof association in telodendrimer solutions at a range of concentrations(72-600 nM) and dissociation in 40 mg/mL of BSA solution is shown inFIG. 4d . The association rate constant (k_(on)) and k_(off) can beobtained by globally fitting the association and dissociation data to a1:1 model algorithm, which gives k_(on)=1.9×10⁴ (±0.8%) M⁻·s⁻¹, andk_(off)=8.1×10⁻⁴ (±0.3%) s⁻¹. The equilibrium binding constant (K_(D))can be therefore calculated by as k_(off)/k_(on), which gives K_(D)=42nM for PEG^(5k)(ArgArg-L-C17)₄ telodendrimer with the dissociationbuffer of BSA solution (40 mg/mL). For the telodendrimers containingeight guanidine groups, the species of hydrophobic moiety slightlyaffect the K_(D) values: C17 groups in the telodendrimer offer strongerbinding with proteins when compared to CHO and VE groups (FIGS. 4d-f ,and Table 3). This fact may relate to the superhigh protein loadingcapacity of C17-containing telodendrimers. For the telodendrimers havingan identical hydrophobic moiety such as CHO, the K_(D) value of the fourguanidine-containing one is much larger than that for the eightguanidine-containing one due to slow association (small k_(on), seeTable 3). It testifies the reduction of the approaching functionalitiesof charged guanidine groups in the telodendrimers causes decrease inassociation rate.

The sizes of protein-loaded telodendrimer nanoparticles slightlydecrease with increasing loading ratio of P/T, as demonstrated by thedynamic light scattering (DLS) studies. The zeta potential of theprotein-loaded telodendrimer nanoparticles generally decreases withincreasing loading ratio of P/T. When P/T mass ratio reaches 1/3, theprotein-loaded telodendrimer nanoparticles have particle sizes of 10-30nm, and neutral zeta potential (<±5 mV), and this P/T mass ratio (1/3)is therefore considered optimal for following studies. Notably, theprotein-loaded telodendrimer nanoparticles have excellent stability, andthey are colloidally stable in PBS during a storage of 2 months at 4° C.(Table 1 and FIG. 15). The sizes of the telodendrimer nanoparticlesbefore and after protein loading are generally stable in a pH range from4.7 to 10. The protein-loaded telodendrimer nanoparticles arenarrow-dispersed and are generally spherical in shape, even for theVE-containing ones. It testifies that the hydrophobic VE groups in thetelodendrimers interact with the hydrophobic residues of the proteinsthat can effectively break the stacking between VE molecules leading tothe disintegration of elongated nanoparticles. Two pieces of evidencemay guide us to understand the binding model of telodendrimer coatingson proteins by reassembly of the telodendrimer micelles (“encapsulationmodel” highlighted by a dotted square in FIG. 1 (left), but not themodel of proteins absorbed on the surfaces of telodendrimer micelles(“absorption model” in FIG. 1 (right): (1) The telodendrimernanoparticle sizes decrease after loading of proteins. (2) A structuralreconstruction occurs for some telodendrimer nanoparticles after proteinloading.

The cooperation of the multivalent charge interactions and hydrophobicinteractions between telodendrimers and proteins are considered to becrucial for effective protein encapsulation. To test this hypothesis, apositively charged telodendrimer with N-acetylated polyarginines andwithout hydrophobic groups (named as PEG^(5k)Arg₄AA₄), and atelodendrimer of PEG^(5k)(Arg(Pbf)-L-CHO)₄ with hydrophobic CHO groupsand without charged groups (guanidine groups in the telodendrimer wereprotected by Pbf protecting groups) were used as controls to compare theprotein loading behaviors with PEG^(5k)(Arg-L-CHO)₄. The chemicalstructures of these telodendrimers are displayed in FIG. 10. The agarosegel retention assay indicated that single type of non-covalentinteraction of either electrostatic or hydrophobic interaction was notefficient to encapsulate or retain the proteins in the telodendrimernanoparticles under electric field. OnlyPEG^(5k)(Arg-L-CHO)₄telodendrimer with dual functionalities highlightedby a dotted square could retard proteins from migration along electricfield, indicating loaded in the nanoparticles with bigger sizes andneutral charges (FIG. 5a ). Notably, the delayed migration of FITC-BSAconjugated with PEG^(5k)Arg₄AA₄ was because of the electrostaticinteractions in the complex that hindered the separation of proteins andtelodendrimers with opposite charges under electric field. Three othertelodendrimers with hydrophobic CHO groups and without charged groups(chemical structures displayed in FIG. 10—PEG^(5k) CHO₈,PEG^(5k)CA₄CHO₄, PEG^(5k)(CA₄-L-CHO₄) were also used in the agarose gelretention assay, and they showed similar migration behaviors withPEG^(5k)(Arg(Pbf)-L-CHO)₄. BLI was employed to further investigate theroles of charged and hydrophobic moieties in the telodendrimers forprotein loading. As shown in FIG. 5b , PEG^(5k)(Arg-L-CHO)₄ with dualfunctionalities triggered a fast association (Table 3). We suggest thatthe fast association is related to both of the charged and hydrophobicmoieties that serve an approaching function for protein capture and playan annealing role to stabilize the captured proteins in thenanoparticles, respectively. Without annealing function by hydrophobicgroups, the telodendrimer having only charged moiety was unable tostably bind to proteins on the sensors mainly due to the highly dynamicassociation-dissociation (FIG. 5c ). On the other hand, thetelodendrimer with only hydrophobic groups had a slow association ratedue to the lack of approaching functional groups, and the associationwas not efficient at low telodendrimer concentrations (FIG. 5d and Table3). The binding between proteins and the telodendrimers with onlyhydrophobic groups might be overestimated by BLI study due to thepresence of hydrophobic interactions between uncoated sensor surfacesand the telodendrimers, since no obvious FRET was observed when thetelodendrimer with only hydrophobic groups was added into the mixture ofRB-BSA and FITC-BSA (significant FRET was only observed in the systemcontaining the telodendrimers with both charged and hydrophobic groups).In a system of lipid-like nanoparticles, Xu and coworkers also observedthat not only charge-charge interaction but also hydrophobic interactioncontributed to the complexation between proteins and lipidoids.

Other polyanionic proteins, such as insulin and green fluorescentprotein (GFP), can also be effectively encapsulated in the telodendrimernanoparticles with a high loading mass ratio of P/T=1/3 (FIGS. 21(a) and(b)). After being loaded in the telodendrimer nanoparticles, the GFPmaintains its fluorescent activity (FIG. 21(c)). This correlates our“green” protein encapsulation approach without organic solvents used.However, positively charged proteins, such as lysozyme, cannot beeffectively loaded in the arginine-containing telodendrimernanoparticles (FIG. 21(d)). This indicates the charge selectivity of thetelodendrimer nanoparticles for protein loading.

For loading of positively charged proteins, we also designed atelodendrimer architecture by simply replacing the guanidine groups inthe arginine-containing telodendrimers with oxalic acid functionalitiesfor systemic delivery of proteins. The chemical structure and thesynthesis route for the telodendrimers containing eight oxalic acidgroups, PEG^(5k)(OAOA-L-R)₄, are displayed in Schemes 11 and 12,respectively. The characterization of molecular properties and proteinloading behaviors are displayed in FIG. 22. As shown in FIG. 22, thePEG^(5k)(OAOA-L-R)₄ telodendrimers can efficiently load a high amount ofpositively charged protein of lysozyme (100% of the telodendrimer byweight). However, due to the negative charge nature of the oxalic acidgroups in PEG^(5k)(OAOA-L-R)₄ telodendrimers, the negatively chargedprotein of BSA cannot be loaded to the telodendrimer nanoparticlesefficiently.

Moreover, we designed a protein-polycation-telodendrimer system forsystemic/extracellular protein delivery. As shown in FIG. 17, thenegatively charged proteins, such as BSA, firstly formed complex withpolycation, e.g., polyethylenimine (PEI), and PEG^(5k)(OAOA-L-R)₄telodendrimers were used to coat the protein-polycation complex toproduce protein-polycation-telodendrimer nanoparticles. BSA and PEIformed stable complex with a well-defined particle size of 10±3 nm at aprotein to polycation mass ratio of 1:2 (FIG. 24). Large aggregatesformed when the protein to polycation ratio larger than 1:2 while excessPEI existed in the systems at protein to polycation ratios smaller than1:2. Therefore, the protein to polycation ratio of 1:2 is considered tobe the best ratio to form protein-polycation complex, and it will beused for further studies. PEG^(5k)(OAOA-L-CHO)₄ telodendrimers were thenused to coat the protein-polycation complex to reduce the surface chargepotential, which also had promise to reduce cytotoxicity of thepolycation, and to avoid nonspecific phagocytosis by thereticuloendothelium systems in vivo. As shown in FIG. 25, theBSA-PEI-PEG^(5k)(OAOA-L-CHO)₄ nanoparticles at aprotein/polycation/telodendrimer mass ratio of 1:2:2 showed stableprotein loading behaviors, a well-defined particle size of 15±5 nm, andneutral zeta potential. Increasing the telodendrimer amount in thesystem resulted in a leakage of loaded BSA, while zeta potential of thenanoparticles obviously increased when a reduced amount of telodendrimerwas used. These facts indicate the protein/polycation/telodendrimerratio of 1:2:2 is the best ratio to formprotein-polycation-telodendrimer nanoparticles. We herein realize theencapsulation of negatively charged protein by PEG^(5k)(OAOA-L-R)₄telodendrimer with the help of polycation.

Other functional groups, such as the crosslinkable boronic acid/catecholpair, can also be introduced into the telodendrimer system, and theresulting telodendrimer (its chemical structure is shown in FIG. 19) isexpected to serve as a smart and robust coating for the delivery ofinsulin or human growth hormone with the ability to release the cargoproteins in response to mannitol and/or acidic pH values.

Cellular Uptake of Protein-Loaded Telodendrimer Nanoparticles. Greenfluorescent FITC-BSA molecules were used to probe the intracellulartrafficking of proteins without and with telodendrimer nanoparticles.HT-29 colon cancer cells were incubated with free FITC-BSA andFITC-BSA-loaded telodendrimer nanoparticles, and were imaged by confocallaser scanning microscopy (CLSM). As shown in FIG. 6a , free FITC-BSAwithout telodendrimer nanoparticles could hardly enter the cellsspontaneously. Only a small amount of the FITC-BSA molecules loaded inthe nanoparticles of the telodendrimers containing four guanidine groupsat a P/T ratio of 1/3 by weight could be delivered to the cellularinteriors (green fluorescence in FIG. 6b ). This result is consistentwith other studies that arginine-containing platforms with less than sixguanidine groups cannot translocate through cell membranes efficiently.The poor intracellular protein delivery efficiency of the telodendrimerscontaining four guanidine groups may restrict their applications forintracellular delivery, however, this endows them with the potential toserve as nanocarriers for systemic delivery of proteins such as insulin.In contrast, significant cellular uptake and intracellular accumulationof the FITC-BSA molecules, that were loaded in the nanoparticles of thetelodendrimers containing eight guanidine groups at a P/T ratio of 1/3by weight, were observed in the cytoplasm of HT-29 cancer cells (greenfluorescence in FIG. 6c ), indicating the ability of eightguanidine-containing telodendrimer nanoparticles for efficient membranetransport of proteins. We also found the species of the hydrophobicmoieties in the telodendrimers affected their protein deliveryefficiency: the delivery efficiency for CHO- or VE-containingtelodendrimers was higher than that for C17-containing telodendrimersmainly because CHO and VE were more likely to inset into cell membranethat contributed to cellular uptake (FIGS. 6b and 6c ). The cellularuptake behavior of protein-loaded telodendrimer nanoparticles in theglioblastoma multiforme (GBM) cell line of U87 was also investigated,which displayed a similar trend with that in HT-29 cells.

The guanidine group is the strongest organic base to form ionic bridgesin protein, also it can form cation-pi interactions. Polyarginine isable to induce efficient membrane transport due to its interaction withthe phosphate or other anionic moieties on cell surfaces. The guanidinefunctionality in the telodendrimers is expected to offer enhancedmembrane transport of proteins. To confirm importance of the guanidinefunctionality, the arginines with guanidine groups in the telodendrimerswere replaced by lysines with amino groups. The chemical structures ofthe telodendrimers containing amino groups are shown in FIG. 20, andtheir characterizations are displayed in FIGS. 23 and 16. Thetelodendrimers containing amino groups have comparable protein loadingcapacities with the telodendrimers containing guanidine groups (FIG.26). However, the telodendrimers containing eight amino groups candeliver significantly less proteins to cellular interiors when comparedto the telodendrimers containing eight guanidine groups (FIG. 27). Thisaffirms the necessity to introduce guanidine functionality to thetelodendrimers for efficient intracellular delivery of proteins.

Hemolysis and Cytotoxicity. The investigation on hemolytic activity isimportant to measure the safety of nanomaterials used for therapeuticdelivery. Our telodendrimer nanoparticles are generally inert in thehemolytic assay (FIG. 28), which indicates safe use for systemicadministration. Only PEG^(5k)(ArgArg-L-C17)₄ shows a slight hemolyticactivity after a long incubation time of 24 h at high telodendrimerconcentrations.

Therapeutic proteins cause cellular cytotoxicity by different pathwaysafter internalization with a potent antitumor effect. However, theunsatisfied cell-penetration abilities of most proteins may limit theirclinical application. Our telodendrimer nanoparticles are able toeffectively deliver proteins across tumor cell membranes, resulting indesire tumor cell apoptosis. Truncated diphtheria toxin (DT₃₉₀), thatinduces cell death by inhibiting protein synthesis after entering intothe cell cytoplasm, was used as a model therapeutic protein toinvestigate the intracellular delivery ability of the telodendrimernanoparticles. The GBM cell line of U87 was exposed to free DT₃₉₀ andDT₃₉₀-loaded telodendrimer nanoparticles for apoptotic analysis. Asshown in FIG. 7, free DT₃₉₀ show nontoxic against U87 cells all throughthe concentrations tested. This is because DT₃₉₀ is unable to enter intothe cell interiors spontaneously. The DT₃₉₀ loaded in the telodendrimernanoparticles containing four guanidine groups (1/3 of P/T by weight)exhibits obvious cytotoxicity only at a high protein concentration of150 nM due to the inefficient cell-penetration. DT₃₉₀ loaded inPEG^(5k)(Arg-L-CHO)₄ and PEG^(5k)(Arg-L-VE)₄ exhibits higher toxicitiesthan C17-containing formulation, which was correlated with the celluptake results (FIG. 7). The bioactive DT₃₉₀ loaded in the telodendrimernanoparticles containing eight guanidine groups (1/3 of P/T by weight)can be potently delivered into the cytoplasm of U87 cells resulting in aprotein-concentration-dependent killing of these cells, and thehalf-maximal growth inhibitory concentration (IC₅₀) values are 32, 34,and 33 nM for DT₃₉₀-loaded PEG^(5k)(ArgArg-L-C17)₄,PEG^(5k)(ArgArg-L-CHO)₄, and PEG^(5k)(ArgArg-L-VE)₄ nanoparticles,respectively. The telodendrimer concentration range used for DT₃₉₀delivery study is 0.3-18.6 μg/mL, and no nanoparticle-relatedcytotoxicity is exhibited in this concentration range for both U87 andHT-29 cell lines. A DTEGF fusion toxin consisting of a truncateddiphtheria toxin, a seven-amino-acid linker, and a human epidermalgrowth factor that can target to U87 cells, was also able to be loadedin the telodendrimer nanoparticles, as determined by an agarose gelretention assay. The DTEGF fusion toxins loaded in the telodendrimernanoparticles had similar IC₅₀ values with that for free DTEGF on U87cells, suggesting that protein bioactivity can be completely maintainedin the arginine-containing telodendrimer nanoformulations.

Our design of telodendrimers with both charged and hydrophobic moietiesis critical for stable and engineerable protein encapsulation andintracellular delivery. On the one hand, sufficient amounts of chargedguanidine groups in the telodendrimers serve an approaching functionthat not only contributes to the rapid/stable protein encapsulation butalso enables efficient membrane transport of proteins. On the otherhand, the hydrophobic groups in the telodendrimers serve an annealingfunction to stabilize the loaded proteins in the telodendrimernanoparticles and various telodendrimers can be produced using diversehydrophobic functional molecules according to different requirements,i.e., C17 molecules are able to endow the telodendrimer nanoparticleswith high protein loading capacity and binding affinity, and CHO- orVE-containing telodendrimers possess satisfied intracellular proteindelivery efficiency. The hydrophobic natural compounds that can be usedfor telodendrimer construction are not limited to these three moleculesreported in this study. The present telodendrimer architecture is aninteresting model to investigate the protein-polymer complexationprocess in detail. We are currently attempting to integrate thistelodendrimer architecture with optimal building blocks selected byvirtual screening of a library of small molecules based on a proteinstructure for protein-specific nanocarrier design.

The facile strategy presented in this study to create telodendrimernanocarriers with multivalent hybrid functionalities is versatile,biologically benign, and relatively inexpensive. We believe that ourtelodendrimer design principle provides useful information to guide thebottom-up rational fabrication of nanocarriers and promote thedevelopment in encapsulation and delivery of protein therapeutics.

Materials. Monomethylterminated poly(ethylene glycol) monoaminehydrochloride (MeO-PEG-NH₂.HCl, Mw: 5 kDa) was purchased from JenkemTechnology. (Fmoc)Lys(Fmoc)-OH and Fmoc-Arg(Pbf)-OH were obtained fromAnaSpec Inc. Cholesteryl chlorofomate was purchased from Alfa Aesar.CellTiter 96® AQ_(ueous) MTS reagent powder was purchased from Promega.Heptadecanoic acid was purchased from Acros. Lysozyme (M_(w) 14.3 kDa,isoelectric point 11.0) was purchased from MP Biomedicals, LLC. Cy5NSsuccinimidyl ester was purchased from AAT Bioquest, Inc. Diisopropylcarbodimide (DIC), N-hydroxybenzotriazole (HOBt), D-α-tocopherolsuccinate, trifluoroacetic acid (TFA), fluorescein isothiocyanate isomerI (FITC), bovine serum albumin (BSA, M_(w) 66.5 kDa, isoelectric point5.4), acetic anhydride (AA), N-hydroxysuccinimide (HOSu), Rhodamine B(RB), and other chemical reagents were purchased from Sigma-Aldrich.Dialysis membrane with 3,500 Mw cut off was purchased from SpectrumLaboratories, Inc. Bovine insulin (M_(w) 5.8 kDa, isoelectric point 5.7)was purchased from Gemini Bio-Products. Recombinant green fluorescentprotein (GFP, M_(w) 28.2 kDa, isoelectric point 6.0) was provided byProf. Stewart N. Loh of Department of Biochemistry and Molecular Biologyat State University of New York Upstate Medical University. Truncateddiphtheria toxin (DT₃₉₀, M_(w) 42.3 kDa, isoelectric point 5.1) andDTEGF (a construct consisting of a truncated diphtheria toxin, aseven-amino-acid linker, and a human epidermal growth factor, M_(w) 49.2kDa, isoelectric point 4.9) were offered by Dr. Walter A. Hall ofDepartment of Neurosurgery at State University of New York UpstateMedical University. The sequences of DT₃₉₀ and DTEGF are listed asfollows:

DT₃₉₀ Sequence: Black = truncated diphtheria toxin(black⇒)GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTNVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKREGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQP FDTEGF Sequence: Black = truncated diphtheria toxin Green = linkerPurple = human epidermal growth factor(black⇒)GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTNVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKREGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPF(green⇒)EASGGPE(purple⇒)NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR

Telodendrimer Synthesis. The telodendrimers with four guanidine groupscontaining heptadecanoic acids, cholesterols and D-α-tocopherolsuccinates, which are named as PEG^(5k)(Arg-L-C17)₄,PEG^(5k)(Arg-L-CHO)₄ and PEG^(5k)(Arg-L-VE)₄, respectively, weresynthesized using a solution-phase condensation reaction starting fromMeO-PEG-NH₂.HCl (5 kDa) via stepwise peptide chemistry. The procedurewas performed as follows: (Fmoc)Lys(Fmoc)-OH (3 eq.) reacted with the Nterminus of PEG using DIC and HOBt as coupling reagents until a negativeKaiser test result was obtained, indicating completion of the couplingreaction. PEGylated molecules were precipitated through the addition ofthe cold ether and then washed with cold ether twice. Fmoc groups wereremoved by the treatment with 20% (v/v) 4-methylpiperidine indimethylformamide (DMF), and the PEGylated molecules were precipitatedand washed three times by cold ether. White powder precipitate was driedunder vacuum. One coupling of (Fmoc)Lys(Fmoc)-OH and one coupling ofFmoc-Arg(Pbf)-OH were carried out respectively upon the removal of Fmocgroups to generate an intermediate of dendritic poly(amino acid)terminated with four Pbf groups and four Fmoc groups on one end of PEG.Then four PEG linker molecules (Mw: 470) were coupled to the aminogroups upon the removal of Fmoc groups with 20% (v/v) 4-methylpiperidinein DMF. After the removal of Fmoc groups, the polymers were coupled withheptadecanoic acid, cholesteryl chlorofomate, or D-α-tocopherolsuccinate. The Pbf protecting groups were consecutively removed via thetreatment with 50% TFA in dichloromethane (DCM) to yieldPEG^(5k)(Arg-L-C17)₄, PEG^(5k)(Arg-L-CHO)₄ and PEG^(5k)(Arg-L-VE)₄ (FIG.8). Positive Kaiser test results were obtained for thesearginine-containing telodendrimers, indicating the successful removal ofPbf protecting groups from the guanidine groups. These resultingtelodendrimers were dissolved in deionized water, dialyzed againstdeionized water for 2 days, and then dried by lyophilization. Thesynthesis procedure of the telodendrimers with eight guanidine groupscontaining heptadecanoic acids, cholesterols and D-α-tocopherolsuccinates, which are noted as PEG^(5k)(ArgArg-L-C17)₄,PEG^(5k)(ArgArg-L-CHO)₄ and PEG^(5k)(ArgArg-L-VE)₄, respectively, issimilar with that for the telodendrimers with four guanidine groups, theonly difference is to couple Fmoc-Arg(Pbf)-OH to the amino groups of thearginines before coupling the PEG linker molecules (FIG. 9). Thesynthesis procedure for the telodendrimers with eight amino groupscontaining heptadecanoic acids, cholesterols and D-α-tocopherolsuccinates (noted as PEG^(5k)(LysLys-L-C17)₄, PEG^(5k)(LysLys-L-CHO)₄and PEG^(5k)(LysLys-L-VE)₄, respectively) is similar with that for thetelodendrimers with eight guanidine groups, the only difference is tocouple twice Fmoc-Lys(Boc)-OH to the amino groups of the polylysinebefore coupling the PEG linker molecules.

The telodendrimer with guanidine groups and without hydrophobic groups(named as PEG^(5k)Arg₄AA₄) was synthesized from acetic anhydride and achemical intermediate 4 in FIG. 8. The Fmoc groups of chemicalintermediate 4 were first removed by the treatment with 20% (v/v)4-methylpiperidine in DMF, and the polymer was then coupled with aceticanhydride using triethylamine as a deacid reagent. The Pbf protectinggroups were consecutively removed via the treatment with 50% TFA in DCMto yield PEG^(5k)Arg₄AA₄.

The telodendrimers containing cholesterol and/or cholic acid groups(named as PEG^(5k)CHO₈, PEG^(5k)CA₄CHO₄, and PEG^(5k)CA₄-L-CHO₄,respectively) were also synthesized using a solution-phase condensationreaction starting from MeO-PEG-NH₂.HCl (5 kDa) via stepwise peptidechemistry. The synthesis and characterization of these telodendrimershave been reported separately¹⁴ and, therefore, are not repeated here.

Determination of Critical Micelle Concentration (CMC). Telodendrimerswere dissolved in phosphate buffered saline (PBS, 1×). The initialmicelle solution was diluted with PBS to obtain the required solutionsranging from 0.39 to 200 μg/mL. A known amount of Nile red in methanolwas added to a series of vials. After methanol was evaporated undervacuum, a measured amount of polymer solutions were added to each vialto obtain a final Nile red concentration of 1 μM. The mixture solutionswere left to shake overnight in the dark and at room temperature. Thefluorescence emission intensity was measured using a microplate reader(Synergy 2, BioTek Instruments, Inc.) at the wavelength of 620 nm withexcitation at 543 nm. CMCs were determined at the intersection of thetangents to the two linear fitting of the curve of the fluorescenceintensity as a function of the log concentration of the telodendrimers.

Encapsulation of Proteins in Telodendrimer Nanoparticles. The proteinsor protein mixtures were dissolved in PBS (1×), and thearginine-containing telodendrimers in PBS (1×) were quickly added intoprotein solution. The proteins were encapsulated by the telodendrimersthrough electrostatic interaction, hydrogen bonding, andhydrophobic-hydrophobic interaction.

Fluorescently Labeled Proteins and Telodendrimers. FITC-labeled BSA(named as FITC-BSA) was synthesized as follow: FITC-BSA was prepared bymixing 3 mg of FITC dissolved in 0.3 mL of DMSO with 10 mL of BSAaqueous solution (10 mg/mL) in the presence of 0.1 M of NaHCO₃ understirring. The molar ratio of FITC to BSA is approximately 5:1. After 24h, the reaction mixture was dialyzed against deionized water in the darkfor one week to remove the unreacted FITC molecules, and dried bylyophilization. FITC-labeled insulin (noted as FITC-insulin) wassynthesized as follow: FITC was dissolved in acetone (1 mg in 200 μL)and added dropwise to a 2.0 mL solution containing the appropriateamount of insulin dissolved in PBS (1×) contained 200 μM EDTA. After 24h, the reaction mixture was dialyzed against deionized water in the darkfor one week to remove the unreacted FITC molecules, and dried bylyophilization. FITC-labeled lysozyme (noted as FITC-lysozyme) wasprepared by mixing 0.5 mg of FITC dissolved in 0.1 mL of DMSO with 15 mLof 3.3 mg mL⁻¹ of lysozyme in PBS (1×). The molar ratio of FITC tolysozyme is approximately 0.4:1. After 24 h, the reaction mixture wasdialyzed against deionized water in the dark for one week to remove theunreacted FITC molecules, and dried by lyophilization. RB-labeled BSA(named as RB-BSA) was synthesized as follows: RB-OSu was firstsynthesized by mixing 10 mg of RB, 3 mg of HOSu and 4 μL of DICdissolved in 0.5 mL of DMSO. RB-BSA was then prepared by mixing aboveRB-OSu solution with 20 mL of BSA aqueous solution (13 mg/mL) in thepresence of 0.1 M of NaHCO₃ under stirring. After 24 h, the reactionmixture was dialyzed against deionized water in the dark for one week toremove the unreacted RB molecules, and dried by lyophilization.FITC-labeled PEG^(5k)(ArgArg-L-C17)₄ (named asFITC-PEG^(5k)(ArgArg-L-C17)₄) was prepared by mixing 0.4 mg of FITCdissolved in 0.2 mL of DMSO with 1 mL of PEG^(5k)(ArgArg-L-C17)₄ aqueoussolution (10 mg/mL) in the presence of 0.1 M of NaHCO₃ under stirring.After 24 h, the reaction mixture was dialyzed against deionized water inthe dark for one week to remove the unreacted FITC molecules, and driedby lyophilization. Cy5-labeled BSA (named as Cy5-BSA) was prepared bymixing 5 mg of Cy5NS succinimidyl ester dissolved in 0.3 mL of DMSO with10 mL of BSA aqueous solution (10 mg/mL) in the presence of 0.1 M ofNaHCO₃ under stirring. The molar ratio of dye to BSA is approximately5:1. After 24 h, the reaction mixture was dialyzed against deionizedwater in the dark at 4° C. for one week to remove the unreacted dyemolecules.

Agarose Gel Retention Assay. Samples in loading buffer (30% glycerolaqueous solution) were loaded into agarose gel (1.5% wt) inTris-acetate-EDTA (TAE) buffer (1×). The gel tray was run for 2 h at aconstant current of 20 mA. The gel was then stained with 1% Coomassieblue (30 min) followed by overnight destaining. The gel was imaged by aBio-Rad Universal Hood II Imager (Bio-Rad Laboratories, Inc.) under SYBRGreen and Coomassie blue modes. The loading capacity and loadingefficiency of the nanoparticles were calculated from the Adj. Vol.(Int.) of the fluorescence bands for unloaded FITC-labeled proteinsusing the Image Lab 3.0 software.

Isothermal Titration calorimetry (ITC). ITC was performed on VP-ITC(MicroCal, LLC) with 1.4 mL cell at 295 rpm stirring at 37° C.Titrations were performed by injecting BSA solution (137 μM) into thecalorimetric sample cell containing 30 μM of telodendrimers using 1 stepof 1.5 μl injection and another 30 steps of 5 μl injections and 300 secpauses between injections to allow the solution to reach equilibrium.Titration of protein into blank buffer (PBS, 1×) was performed forreference. Protein sample was dialyzed against buffer solution (PBS, 1×)for two days before the ITC measurement, and the concentration wascalculated from the UV-vis spectrum based on the molar extinctioncoefficient of 43,824 M¹ cm¹ for BSA at 279 nm. Heats of injections werecalculated using Microcal analysis package for Origin 7.0.

Förster Resonance Energy Transfer (FRET) Studies. RB-BSA andFITC-PEG^(5k)(ArgArg-L-C17)₄ were used to prepare FRET nanoparticles.Equal volumes of 2 mg/mL of FITC-PEG^(5k)(ArgArg-L-C17)₄ solution and 2mg/mL of RB-BSA solution were mixed, following by stirring overnight.Concentrated BSA solutions were then added into the above mixtures toreach final BSA concentrations from 0 to 40 mg/mL. The fluorescencespectra with a range from 480 to 640 nm at different time points excitedby 439 nm were recorded using a microplate reader (BioTek Synergy 2).The FRET ratio was calculated by the formula of [100%×I₅₈₄/(I₅₈₄+I₅₂₈)],where I₅₈₄ and I₅₂₈ were fluorescence intensities of RB-BSA at 584 nmand FITC-PEG^(5k)(ArgArg-L-C17)₄ at 528 nm, respectively. FITC-BSA andRB-BSA were also mixed for FRET study.

Bio-Layer Interferometry (BLI). The binding affinities of telodendrimersto proteins were measured at 37° C. by BLI on an Octet-Red 96(ForteBio). Streptavidin biosensors (ForteBio) were prewetted in 40mg/mL of BSA solution for 900 s, and incubated in the same solution for900 s, washed in PBS for 480 s, and transferred to wells containingtelodendrimers at concentrations ranging from 75 to 600 nM in PBS for900 or 1800 s (association). Dissociation at each studied concentrationwas carried out in either PBS (1×) or BSA solution (5 or 40 mg/mL) for1800 s. The k_(on) and k_(off) values were obtained by fitting theassociation and dissociation data to a 1:1 model algorithm using Octetsoftware. The K_(D) derived from kinetic fitting was calculated ask_(off)/k_(on).

Hemolytic Assays. One milliliter of fresh blood from healthy humanvolunteers was collected into 5 mL of PBS solution in the presence of 20mM EDTA. Red blood cells (RBCs) were then separated by centrifugation at1000 rpm for 10 min. The RBCs were washed three times with 10 mL of PBSand resuspended in 20 mL of PBS. Diluted RBC suspension (200 μL) wasmixed with nanoparticle PBS solutions at serial concentrations (10, 100,and 500 μg/mL) by gentle vortex and incubated at 37° C. After 0.5 h, 4h, and over night, the mixtures were centrifuged at 1,000 rpm for 5 min.The supernatant free of hemoglobin was determined by measuring theabsorbance at 540 nm using a UV-vis spectrometer. Incubations of RBCswith Triton-100 (2%) and PBS were used as the positive and negativecontrols, respectively. The percent hemolysis of RBCs was calculatedusing the following formula:

$\begin{matrix}{{{RBC}\mspace{14mu}{hemolysis}} = {\frac{\left( {{OD}_{sample} - {OD_{neg{ative}\mspace{14mu}{control}}}} \right)}{\left( {{OD}_{{positive}\mspace{14mu}{control}} - {OD_{{negative}\mspace{14mu}{control}}}} \right)} \times 100\%}} & (1)\end{matrix}$

Cell Culture and MTS Assays. The human glioblastoma multiforme cell lineU87 and the colon cancer cell line HT-29 were purchased from AmericanType Culture Collection (ATCC, Manassas, Va., U.S.A.). All cells werecultured in 100 U/mL penicillin G, and 100 μg/mL streptomycin at 37° C.using a humidified 5% CO₂ incubator. Various formulations of proteinswith different dilutions were added to the plate and then incubated in ahumidified 37° C., 5% CO₂ incubator. After 4 h incubation, McCoy's 5Amedium supplemented with 10% fetal bovine serum (FBS) was added into theabove system. After 72 h incubation, a mixture solution composed ofCellTiter 96 AQ_(ueous) MTS, and an electron coupling reagent, PMS, wasadded to each well according to the manufacturer's instructions. Thecell viability was determined by measuring absorbance at 490 nm using amicroplate reader (BioTek Synergy 2). Untreated cells served as thecontrol. Results were shown as the average cell viability[100%×(OD_(treat)−OD_(blank))/(OD^(control)−OD_(blank))] of triplicatewells. The cells were also treated with blank nanoparticles in PBS atdifferent dilutions and incubated for a total of 72 h to evaluatenanoparticle-related toxicity.

Cellular Uptake. The cellular uptake and intracellular trafficking ofthe protein-incorporated nanoparticles were determined by fluorescencemicroscopy. FITC-BSA was used as a model protein. HT-29 and U87 cellswere seeded in chamber slide with a density of 5×10⁴ cells per well in350 μL of McCoy's 5A and cultured for 24 h. The original medium wasreplaced with free FITC-BSA and FITC-BSA-loaded nanoparticles at a finalFITC concentration of approximately 1.5 μg/mL at 37° C. After a 3 hincubation, the cells were washed three times with cold PBS (1×) andfixed with 4% formaldehyde for 10 min at room temperature, and the cellnuclei stained with DAPI (blue). The slides were mounted with coverslips and cells were imaged with a NiKON FV1000 laser scanning confocalfluorescence microscope.

Characterization. Proton NMR spectrum was recorded on a Bruker AVANCE600 MHz spectrometer. MALDI-TOF MS spectrum was recorded on a BrukerREFLEX-III instrument. Dynamic light scattering (DLS) studies wereperformed using a Zetatrac (Microtrac Inc.) instrument, and thearea-based mean particle sizes were presented. Zeta potentialmeasurements were carried out on a Malvern Nano-ZS zetasizer at roomtemperature. UV-vis spectra were recorded on a Thermo ScientificNanodrop 2000c spectrophotometer. TEM images were taken on a JEOLJEM-2100 HR instrument operating at a voltage of 200 kV. The sampleswere prepared by dropping the solutions onto carbon coated grids, andstained by uranyl acetate.

TABLE 1 Properties of the telodendrimers before and after loading ofproteins. zeta D_(h) (nm) potential D_(h) with zeta (mV) (nm) proteinM_(w) M_(w) potential D_(h) CMC with with after telodendrimer(Theo.)^(a) (MS)^(b) (mV)^(c) (nm)^(c) (μM)^(d) protein^(e) protein^(e)storage^(f) PEG^(5k)(Arg-L-C17)₄ 7,940 7,941 −4.6 ± 0.7 11 ± 3 2.68 −5.1± 0.7 10 ± 4 11 ± 3 PEG^(5k)(Arg-L-CHO)₄ 8,581 8,314 −4.1 ± 0.5 27 ± 81.32 −4.3 ± 0.3 22 ± 7 15 ± 5 PEG^(5k)(Arg-L-VE)₄ 8,981 8,728 −3.2 ± 1.225 ± 9 1.35 −4.4 ± 0.3 19 ± 9 22 ± 8 PEG^(5k)(ArgArg-L-C17)₄ 8,565 8,599−2.4 ± 0.6 17 ± 4 1.45 −3.4 ± 0.3 13 ± 4 14 ± 4 PEG^(5k)(ArgArg-L-CHO)₄9,206 9,231 −4.0 ± 0.1 18 ± 4 1.38 −4.4 ± 0.1 12 ± 4 15 ± 5PEG^(5k)(ArgArg-L-VE)₄ 9,606 9,624 −1.2 ± 0.8  32 ± 13 1.14 −3.0 ± 0.618 ± 8 27 ± 9 ^(a)Theoretical molecular weight. ^(b)Acquired byMALDI-TOF MS analysis. ^(c)Obtained in PBS at a concentration of 1mg/mL. ^(d)Measured by fluorescent method using nile red as a probe.

TABLE 2 Summary of isothermal titration calorimetry results.telodendrimer protein (equiv)^(a) K_(a) (M⁻¹) ΔH (kcal · mol⁻¹) ΔS (cal· mol⁻¹ · K⁻¹) PEG^(5k)(ArgArg-L-C17)₄ 0.11 1.1 × 10⁶ 22 100PEG^(5k)(ArgArg-L-CHO)₄ 0.15 1.8 × 10⁵ −37 −96 PEG^(5k)(ArgArg-L-VE)₄0.09 3.5 × 10⁵ −34 −84 ^(a)BSA equivalents conjugated withtelodendrimers.

TABLE 3 Summary of bio-layer interferometry results. telodendrimer K_(D)(nM) k_(on) (M⁻¹ · s⁻¹) k_(off) (s⁻¹)^(b) PEG^(5k)(ArgArg-L-C17)₄ 42 1.9× 10⁴ (±0.8%) 8.1 × 10⁻⁴ (±0.3%) PEG^(5k)(ArgArg-L-CHO)₄ 49 1.7 × 10⁴(±0.7%) 8.5 × 10⁻⁴ (±0.3%) PEG^(5k)(ArgArg-L-VE)₄ 59 7.8 × 10³ (±0.6%)4.6 × 10⁻⁴ (±0.3%) PEG^(5k)(Arg-L-CHO)₄ 89 7.9 × 10³ (±0.6%) 7.0 × 10⁻⁴(±0.4%) PEG^(5k)Arg₄AA₄ ^(a) N/A N/A N/A PEG^(5k)(Arg(Pbf)-L-CHO)₄ 883.3 × 10³ (±0.6%) 2.9 × 10⁻⁴ (±0.4%) ^(a)No obvious association wasobserved in the telodendrimer concentration range of 75-600 nM. ^(b)BSAsolution (40 mg/mL) was used as the dissociation buffer.

1. A compound of formula (I):

wherein PEG is optionally present and is a polyethylene glycol moiety,wherein PEG has a molecular weight of 44 Da to 100 kDa; X is optionallypresent and is a branched monomer unit; each L¹ is independentlyoptional and is a linker group; each L² is independently optional and isa linker group; each L³ is independently optional and is a linker group;each L⁴ is independently optional and is a linker group; D¹ is optionaland is a dendritic polymer moiety having one or more branched monomerunits (X), and a plurality of end groups; D² is a dendritic polymerhaving one or more branched monomer units (X), and a plurality of endgroups; R¹ is optional and is an end group of the dendritic polymer andis independently at each occurrence in the compound selected from thegroup consisting of crosslinkable groups; R² is an end group of thedendritic polymer and is independently at each occurrence in thecompound selected from the group consisting of positively or negativelycharged groups and neutral groups (e.g., polar groups: sugars, peptides,hydrophilic polymers, or hydrophobic groups: long-chain alkanes (C₁-C₅₀)and fatty acids (C₁-C₅₀), aromatic molecules, esters, halogens,nitrocompounds, anthracyclines, fluorocarbons, silicones, certainsteroids such as cholesterol, terpenoids, vitamins, and polymers, andamphiphilic groups, cholic acid, riboflavin, chlorogenic acid), where atleast one positively or negatively charged groups are present in R²;subscript x is an integer from 1 to 64, wherein subscript x is equal tothe number of end groups on the dendritic polymer; subscript y is aninteger from 1 to 64, wherein subscript y is equal to the number of endgroups on the dendritic polymer; subscript p is an integer from 0 to 32;and subscript m is an integer from 0 to
 32. 2. The compound of claim 1,wherein at each occurrence in the compound the branched monomer unit (X)is independently selected from the group consisting of a diaminocarboxylic acid moiety, a dihydroxy carboxylic acid moiety, and ahydroxyl amino carboxylic acid moiety.
 3. The compound of claim 2,wherein at each occurrence in the compound the diamino carboxylic acidis independently selected from the group consisting of 2,3-diaminopropanoic acid, 2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid(ornithine), 2,6-diaminohexanoic acid (lysine), (2-Aminoethyl)-cysteine,3-amino-2-aminomethyl propanoic acid, 3-amino-2-aminomethyl-2-methylpropanoic acid, 4-amino-2-(2-aminoethyl) butyric acid, and5-amino-2-β-aminopropyl) pentanoic acid.
 4. The compound of claim 2,wherein the diamino carboxylic acid moiety is an amino acid moiety. 5.The compound of claim 1, wherein each branched monomer unit X is lysinemoiety.
 6. The compound of claim 1, wherein the compound is selectedfrom the group consisting of:

wherein each branched monomer unit is individually selected from alysine moiety and arginine moiety.
 7. The compound of claim 1, whereinat each occurrence in the compound the linker L¹, L², L³ and L⁴ each areindependently selected from the group consisting of a polyethyleneglycol moiety, polyserine moiety, enzyme cleavable peptide moiety,disulfide bond moiety and acid labile moiety, polyglycine moiety,poly(serine-glycine) moiety, aliphatic amino acid moieties, 6-aminohexanoic acid moiety, 5-amino pentanoic acid moiety, 4-amino butanoicacid moiety, and beta-alanine moiety.
 8. The compound of claim 1,wherein at each occurrence in the compound the linker L¹, L², L³ and L⁴are independently selected from the group consisting of:


9. The compound of claim 1, wherein the linker L¹, L², L³, L⁴ or acombination thereof comprises a cleavable group.
 10. The compound ofclaim 9, wherein the cleavable group is a disulfide cleavable moiety.11. The compound of claim 1, wherein the (PEG)_(m) portion of thecompound is selected from the group consisting of:

wherein each K is lysine.
 12. The compound of claim 1, wherein at leastone (e.g., 1 to 128) of the R² groups are charged groups and,optionally, at least one of the R² groups are neutral groups, and,optionally, at least one of (e.g., 1 to 128) of the R¹ groups, ifpresent, are reversible crosslinking groups.
 13. The compound of claim12, wherein the reversible crosslinking group(s) is/are coumarin moiety,4-methylcoumarin moiety, boronic acid moiety or derivative or analogthereof, catechol moiety or derivative or analog thereof, cis-diolmoiety or derivative or analog thereof, cinnamic acid moiety orderivative or analog thereof, chlorogenic acid moiety or derivative oranalog thereof, amine moiety or a derivative thereof, carboxylic acid ora derivative thereof, acyl group, or a derivative thereof, epoxide or aderivative thereof, thiol group or a derivative thereof, malaimide or aderivative thereof, alkene or a derivative thereof, azide or aderivative thereof, alkyne or a derivative thereof, coumarin or aderivative thereof, or a combination thereof.
 14. The compound of claim12, wherein the charged group is the moiety or derivative or analog ofarginine, lysine, guanidine, amine, amidine, tetrazole, hydroxyl,carboxyl, phosphate, sulfonate, methanesulfonamide, sulfonamide, oroxalic acid and functional groups.
 15. The compound of claim 12, whereinthe neutral group is the moiety or derivative or analog of sugars,peptides, hydrophilic polymers, long-chain alkanes (C₁-C₅₀) and fattyacids (C₁-C₅₀), aromatic molecules, esters, halogens, nitrocompounds,anthracyclines, fluorocarbons, silicones, certain steroids such ascholesterol, terpenoids, vitamins, and polymers (e.g., PLGA,polycaprolactone, polylactic acid, polyglycolic acid, polystyrene andpolyisoprene, polyvinyl pyridine); amphiphilic groups, cholic acid,riboflavin, chlorogenic acid and natural compound extract and syntheticcompounds.
 16. A nanocarrier comprising a plurality of compounds ofclaim
 1. 17. The nanocarrier of claim 16, wherein the nanocarrierfurther comprises one or more charged proteins.
 18. The nanocarrier ofclaim 17, wherein the nanocarrier further comprises a cationic polymer.